Indian Journal of Biochemistry & Biophysics Vol. 39, February 2002, pp. 5-15

Minireview

On the importance of backbone loop and peptide flip in the Walker sequence in FI-ATPase action

T Ramasarma*t and C Ramakrishnano

Department of Biochemistry* and Molecular Biophysics Unite, Indian Institute of Science, Bangalore 560 012

Received 16 November 2001

The Walker sequence, GXXXXGKT, present in all the six subunits of FI-ATPase exists in a folded form, known as phosphate-binding loop (P-Ioop). Analysis of the Ramachandran angles showed only small RMS deviation between the nucleotide-bound and nucleotide-free forms . This indicated a good overlap of the backbone loops.

The catalytic /3-subunits (chains D, E and F) showed significant changes in the Ramachandran angles and the side chain torsion angles, but not the structural a-subunits (chains A, B and C). Most striking among these are the changes associated with Vall60 and Glyl61 corresponding to a flip in the peptide unit between them when a nucleotide is bound (chains D or F compared to nucleotide-free chain E) .

The conformational analysis further revealed a hitherto unnoticed hydrogen bond between amide-N of the flipped Gly 161 and terminal phosphate-O of the nucleotide. This assigns a role for this conserved amino acid, otherwise ignored, of making an unusual direct interaction between the peptide backbone of the enzyme protein and the incoming nucleotide substrate. Significance of this interaction is enhanced, as it is limited only to the catalytic subunits, and also likely to involve a mechanical rotation of bonds of the peptide unit. Hopefully this is part of the overall events that link the chemical hydrolysis of ATP with the mechanical rotation of this molecule, now famous as tiny molecular motor.

Introduction Walker and colleagues I first recognized the

sequence of GXXXXGKT as the. nucleotide-binding site in FI-ATPase. It occurs in several proteins that use A TP or GTP as the energy source, and is iden­tified as the location for fitting the phosphate chain in position. It became known as Walker motif A. Crystal structure data of some proteins indicated that this motif is present in the shape of a loop around the phosphate chain of the nucleotide2

-4

. Found in many such nucleotide-binding proteins, the consensus sequenc:e of GXXXXGKT (S), with S instead ·0f T in some 'case$-, is referred as the ' Walker loop or the phosphate-btnding loop (P-loop).

Action of F I-A TPase- begins with the substrate meeting with the polypeptide chain at the Walker sequence. It is here that A TP is bound, its y-phosphate is split and ADP is released. For ready appreciation of trimming off the terminal phosphate here, this was referred as the sickle (P-loop) and hammer (ATP) models. It should be remembered that the same protein in the cell acts in the reverse as ATP synthase,

tCorresponding author Email : trs @biochcm.ii ~c .emct.in ;

Phone: 080-3092538: fax: 080-3601683.

and the true cellular function of this very loqp is in the synthesis £?f ATP. Thus: the Walker sequence assumes signific'ance ' in the 'i~itial ~vents of translation of

_ chemiCal energy into mecbanrcalforin. . There are several proteins with different Walker sequences of the variable quartet (XXXX) (Brook­haven Protein Data Bank6

). An evaluation of Rama­chandran angles7

.8 of the backbone (q,,\jI) and the side­

chain torsion angles (X) of tr.ese"proteins indicated that only those with the property of binding and using nucleotides share a similar conformation and the common loop structure9

. ~nexpected, large vai-iQtions in the Ramachandran angles, on the other hand, were found in some sets of proteins with same Walker sequence. Examination 'of their structures led to the finding that one of their peptide units was in a flipped state'). These fitted with the classical ~_turn s I O. ll except in the case of a su bunit of F I-ATPase, wherein the RamacLandran angles are far different from characteristic ~-turn ranges. In the light of importance of the loop structure of Walker sequence at the site of action for phosphate exchanges, we decided to probe further into structural details of the peptide flip in th is protein hoping to derive clues on its relationship to the function. Our observations are recorded here.

6 INDIAN J. BIOCHEM. BIOPHYS., VOL. 39. FEBRUARY 2002

The subunit assembly in the structure of F.-ATPase

Mitochondrial FI-ATPase has three subunit proteins (a3 ~3 y). These are arranged in the form of a rotary shaft of an unusually long pair of a-helices of the y-subunit within a bulb-like stator structure made up of three alternating segments of a- and ~-subunits. This is delineated by the first crystal structure obtained by Walker and co-workers3

. Named chains A, Band C, the a-subunits bind A TP without turnover and seem to have a structural role. The corresponding ~-subunits, named chains D, E, and F, bind to ADP, no nucleotide (empty) and to ATP, respectively. The six subunits are placed alternatively in the sequence A D BEe F, with the active site situated at the interface of two subunits.

The a- and ~-subunits are structurally related, but have distinctive roles. Only the ~-subunits are catalytically active in ATP hydrolysis. The non­hydrolyzable ATP analogue, AMPPNP, was used in these studies because of high activity of hydrolysis of ATP. The frozen form thus obtained is referred hereafter as ATP form. The outstanding value of the

(A)

(D)

Walker structure3 is its fortuitous possession of the three conformational states of the catalytic cycle in the same protein molecule. Therefore we chose this to probe into the conformational changes that occur in the three states.

The sickle-like loop of Walker sequence in F.-ATPase

This peptide segment of GXXXXGKT occurs at the nucleotide-binding site in all the six subunits (a­subunits, residues 169-176, GDRQTGKT; ~-subunits, residues 156-163, GGAGYGKT) of the protein, FI-ATPase. The line diagrams of the backbone of the polypeptide chains of the six subunits, shown in Fig. 1, confirm the loop structure. These are drawn using the package of RASMOL I2 . The peptide unit spanning residues 5 (TIV) and 6 (G;, is taken as the internal frame of reference. A common sickle-like folding is found in all the six structures. Good overlap of the backbone atoms is anticipated with nearly the same structure for segment 5-8 and a small variation in the bending of the segment 1-4. This is confirmed by the Ramachandran angles.

(B) ee)

(E) (F)

Fig. I-Wire frame diagrams of the backbone atoms in the P-Ioop segments (GXXXXGKT) of F)-ATPase [The residues 169-176 of chains A-C and 156-163 of chains D-F are arranged right to left. Notice the sickle-like folding of all the chains. Variation in the C­terminus is little. Some compactness is sccn in the N-terminus of chains D-F compared to A-C. The significant change is the peptide flip in the chain E with the fifth carbonyl-O (red) pointing inwards compared to the last three oxygen atoms on its left.]

RAMASARMA & RAMAKRISHNAN: WALKER SEQUENCE IN FI-ATPase ACTIO 7

Comparison of the Ramachandran angles of the Walker loop

The data on the Ramachandran angles for the GXXXXGKT segment in the three ATP-bound forms

of ex-subunits (chains A-C, segment 169-176) are

g iven in Table I . Note the largest difference in the

angles between these chains is only 30°, indicating that the backbone folding remains almost invariant.

The corresponding conformati onal angles of the ~­subunits (chains D-F, segment 156-163) are given In

Table I - The Ramachandran angles o f the backbone (<I>,\jI) and of the side chain torsion ang les (X) of residues in the

P-Ioop of a-subunits in FI-ATPase

[The three subun its are identifi ed as cha ins A. Band C. The angles o f <I>,\jI for the segment 169- 176 (P- Ioop) for the three chains are

given. T he side-chain torsion ang les (X) are given only for residues Asp 170. Arg 171 . GIn 172. Thr 173. Lys 175 and Thr 176 as the

others. being glyc ine, have no relevant values. Absolute differences in these angles between the three forms (181) are also g iven. The changes for almost all are small indicating si milar conformation of these chains]

Residue

Gly l 69

Asp l 70

Arg l 7 1

Glnl 72

Thr l 73

G ly l 74

Lys l 75

Thrl76

A spl70

A rg l71

Glnl72

Thr 173

Lys l75

Thrl76

Parameter

<I>

\jI

<I>

\jI

<I>

\jI

<I>

\jI

<I>

\jI

<I>

\jI

<I>

\jI

<I>

\jI

Xl (N-Cu_CII-CY)

X21 (CIl_CB_CY_OSI)

Xl (N _Cu _C II_CY)

X2 (C"-CII-CY-C5)

X' (CI1_CY_C&_Nf)

X4 (CY-C&-N' -C()

X" (CO-N'-C~-N~)

Xl (N_C"_CI1_CY)

X2 (Cu_CI1_CY_Co)

X" (CI1-CY-Co_O£I)

X 32 (CP_CY_CO_N E2)

X" (N-C"-CII-OYI)

XI2 (N _C"_CB_CY2)

Xl (N_CIl_CB_cv)

Xl (Cr1_CB_Cy_c 5)

X3 (CII-CY-CO-NE)

X4 (CY-CS-N £-N~)

X" (N_CIl_CI1_OYI)

XI 2 (N_C" _C I1_Cy2)

Chain A

174

176

-76

- 176

-62

152

43

55 -113

-II

11 8

7

-53

-60

-64

-32 -65

-29

46

- 175

167

77

o -8 1

-69

-48

132

16

- 103

-67

167

-64

140

-54

- 176

Chain B

178

175

-78

- 176

-63

147

55

40

-104

4

93

37

-72

-65

-48

-44

-56

-3 1

56

-169

174

58

o -77

-85

-69

11 0

32 -89

-69

166

-75

138

-56

- 176

Torsion angle. degrees

Chain C

170

172

-72

179

-59

145

59

28

-96

- I

105

15

-55

-50

-6 1

-39

-58

-61

52 180

139

93

o -68

-68

-57

120 42

-76

-57

159

-105

139

-64

177

18(A13)1

4

I

2

o I

5

12

15

9

15 25

30

19

5

16

12

9

2

10

6

7

19

o 4

16

21 22

16

14

2

I

II

2

2

2

18(BC)1

8

3

6

5 4

2

4

12

8

5

12 22

17

15 13

5

2

30

4

II

35

35

o 9

17

12 10

10

13 12

7

30

I

8

7

8(AC)

4

4

4

5

3

7

16

27

17

10

13

8

2

10

3

7

7

32

6

5

28

16

o 13

I

9

12 26

27

10

8

41

I

10

9

8 INDIAN J. BIOCHEM. BIOPHYS., VOL. 39, FEBRUARY 2002

Table 2. The root mean square (RMS) deviations by the best-fit method are found to be 1.57 A for the A TP-form, and 1,52 A for the ADP-form compared to the open form with no nucleotide. The RMS deviation between the two nucleotide-bound forms is even less at 0.46 A, indicating neglig ible variation in backbone folding. This is further confirmed by the observation that differences in torsion angles are less than 20° on loss of terminal phosphate in A TP (see Table 2, last column). Grossly viewed, the folding of the backbone of these P-loop chains is similar and they can overlap on each other (see Fig. 1). Thi s means that the overall structure of the P-loop backbone is unresponsive to binding of ATP and subsequent changes. But there are some changes in the side chains on nuc leotide binding.

Comparison of side chain torsion angles of the Walker loop

There is little difference in the side chain torsion angles of the GDRQTGKT segments in the u­subunits. The highest 181 value is only 35° for the pairs of chains of AB, BC and AC (Table 1). This similarity in structures is expected as all these chains bind to ATP.

Major changes occur in these torsion angles of the GGAGYGKT segments in the B-subunits. The only residues relevant for this purpose are Ya1160, Lys162 and Thr163 , others being glycine or alanine. Comparing the nucleotide-bound forms of chain D and F with the nucleotide-free form of chain E, differences larger that 45° are found (Table 2, see the values shown in box).

Table 2 - The torsion angles of the backbone (<I>,\fI) and of the side-chains (X) of residues in the P-Ioop of B-subuni ts in FI-ATPase

[The three conformational states of ADP-bound, nucleotide-free and ATP-bound forms of B-subunits are identified as chains 0, E. and F, respectively. The Ramachandran angles (<I>,\fI) for the segment 156-163 (P- Ioop) for the three chains are n:corded. The side­chain torsion angles (X) are given only for residues Va1160, Lysl62 and Thr1 63, as the others, being glycine or alanine. have no

relevant values . Absolute differences in these angles between the nucleotide-bound and nucleotide-free forms (DOE and oEF) and between the two nucleotide-bound forms (oDF) are also given. Changes exceeding 40° are shown in box. ]

Residue Parameter Torsion angle, degree

Chain 0 Chain E Chain F lo(DE)1 lo(EF)1 18(DFI

Gly l56 <I> -174 93 -158 § [@2] 16

\fI 145 153 136 8 17 9 Gly l57 <I> -75 - 125 -74 ~ ~ I

\fI -176 -107 - 179 ~ § 3

Alal58 <I> -65 -86 -59 2 1 27 6

\fI 127 159 138 32 2 1 II

Gly l59 <I> 68 78 57 10 21 II

\fI 41 -37 35 ~ § 6

Val 160 <I> -1 12 -86 -98 26 12 14

\fI -8 133 -22 [ill] [ill] 14

Gly l61 <I> 128 -48 139 [jlg [ill] II

\fI 5 30 6 25 24 I

Lys 162 <I> -52 -41 -56 II 15 4

\fI -60 -58 -54 2 4 6 Thr l63 <I> -64 -69 -67 5 2 2

\fI -43 -22 -41 2 1 19 2 Val160 X" (N-C"-CB-CYI ) -77 -83 -55 6 28 22

Xl 2 (N-C"-CB-CY2) 47 39 -173 8 ~ ~ Lysl62 Xl (N-C"-CB-CY) -55 - 164 -51 [Q2l UTI 4

X2 (C"-CB-CY-Co) -1 78 -131 173 ~ ~ 5

X3 (Cfl-CY-Co-C') -85 174 -70 [IQI] m 15

X4 (CY-Co-C'-NC) -47 140 -99 [ill] [ill 52

Thrl63 Xii (N-C"-Cfl-O¥I) -57 80 -62 [ill] [@] 5 Xl 2 (N-C"-CB-C¥2) -1 78 -41 175 [ill] ~ 7

RAMASARMA & RAMAKRISHNAN: WALKER SEQUENCE IN F1-ATPase ACTION 9

All the torsion angles of Lys162 in chain E are in the neighborhood of 150° indicating near extended form placing its N~ pointing away from the backbone. Differences of its angles of Xl, X2

, X4 are large, and of X3 appreciable, once ATP or ADP binds. Being in the neighborhood of -60° (gauche form), the angles of Xl,

X2, X4 ensure bending of the side chain over the

nucleotide and thereby a hydrogen bond between its N~ and terminal phosphate-O. Same trend is shown by the torsion angles Xii and Xl 2 of Thrl63. The gO and g+ conformation of the free form changes to gO and t on binding to nucleotides, while retaining the side chain orientation. This can be attributed to the need to bring its 0\ present in a totally unsuitable form, into a position to coordinate with Mg. All the above mentioned changes are consequent to nucleotide binding as they occur in both A TP and ADP forms.

Differences were observed between the nucleotide forms with respect to two changes [see 18(DF)1 values in the last column in Table 2]. First, the relatively large difference of 52° of torsion angle X4 of Lys 162 indicates movement of its N~ through rotation about Co . . . CY bond, the rotational parameter of X4, to facilitate its reaching the shorter phosphate chain of ADP and form the hydrogen bond. This rotation in the other direction is essential to accommodate ATP. This is judged by steric hindrance of 2.0 A between N~ and the phosphate-O, when introduced geometrically in the same orientation of y-phosphate as in chain F. Second, the torsion angle Xl2 of Val163 shows a significant difference with a rotation of 120° of its side chain between A TP- and ADP-forms. The reason for this structural change that occurs on hydrolysis of A TP is not apparent.

Peptide-flip in the P-Ioop of nucleotide-free ~-subunit - a striking new observation

The P-Ioop structure described above is deceptively stable. On comparing the conformations of the P-Ioop in different states, a striking change unexpectedly became highlighted. Note that the carbonyl-O of Gly 161 of chain E faces the other way in contrast to those of chains D and F, and of other oxygen atoms in the loop (Fig. 1). From an evaluation of the torsion angles (Table 2), it became obvious that this is due to flip of a single peptide unit between Val160 and Gly 161 in chain E compared to chains D and F.

Nucleotide-bound forms of the P-Ioop showed dramatic changes in the torsion angle 'II of Val160 and the torsion angle <l> of Gly 161 with large devia­tions (142-176°, shaded in Table 2) from the corres­ponding values of the nucleotide-free form. This is clear in the Ramachandran map by large shifts highlighted by arrows (Fig. 2). Small RMS deviations between the nucleotide forms of the protein, the major changes occurring on binding of ATP, and their reversal on removal of ADP are reiterated by the data in the Ramachandran map (Fig. 2). Such a large change of'll of a residue and a concomitant change of <l> in the successive residue without altering the overall folding of the chain are characteristic of flip of the peptide unit between the two residues l2

.

Less obvious changes on nucleotide binding in torsion angles can also be noticed in the Rama­chandran map of the ~-subunits (Fig. 2). Changes in the torsion angle'll of Gly157 and Gly159 of about 70° with minimal changes in nearby bonds are also significant (Table 2). This results in a more compact folding by bringing in the segment 160-168 closer to segment 158-159. Changes in the torsion angle <1> of Gly156 (93° and 109°) and Gly157 (50° and 51 °) are considerable, albeit smaller than that of Gly 161 (176° and 173°). There are four glycyl residues out of eight in this P-Ioop. These must be doing something more than simply creating space for transient occupation of the leaving phosphate group. Is it possible that these manifestations are part of primary rotation of the backbone to obtain the flip , and therefore the initial small steps towards mechanical change?

This kind of peptide flip, characteristic of inter­conversion of types I and II of ~-tums l3 , occurs in several proteins with a glycine overwhelmingly at position i + i 2

• Compensatory rotation of bonds on the two sides of this peptide unit in steps to change the torsion angles , but retaining the backbone folding, requires energy and this was computed in ~-tum to be about 3 kcallmole for a single bond rotation l2

.

To the best of our knowledge, flipping of the peptide unit between Val160 and Gly 161 in the ~­

subunit of FI-ATPase had not been mentioned anywhere. While describing the nucleotide-binding sites in the classic paper on the subject3

, the backbone was simply recorded as holding the side chains with hardly any clarity on the peptide units and the direction they point. The distances between N

10 INDIAN J. BIOCHEM. BIOPHYS., VOL. 39, FEBRUARY 2002

180

-180

o ~ , , ,

0

~''<I-

, , , ,

*A 58

YJ

O~4

63

tt- 57

~------ *G156 -- +

--\ \ 6161*+

180

--~~~ 0

-180

Fig. 2-The Ramachandran plots of the P-Ioop of chains D, E and F in FI-ATPase. [The plots of conformation of the eight residues in the segment 156-163 in the (cjHJI) pl ane superposed on the allowed regions for L-residue are shown for the chain D (ADP bound; *), the chain E (empty nucleotide-free ; 0) and the chain F (ATP-bound; +). Thin , broken arrows indicate shifts in the co nformatio nal points on loss of bound ATP or ADP. The po ints for G 156 that lie in the top left quadrant are shown outside the top right quadrant to connect them by the arrow with their nuc leotide-free form. Notice the appreciable differences in torsion angles of IJI of VI60 and ¢ of GI61 on loss of nucleotide. characteristics of peptide flip, shown by the long thick, broken arrows. l

(161) .. . 0IB and the angles C (160)-N (161)-0IB for ADP-form (2.88 A and 125°) and for ATP-form (3 .04 A and 126°) are consistent with presence of strong hydrogen bonds (Table 3) . Without this flip , thi s crucial hydrogen bond between ~-phosphate-O and amide-N ofGly161 is not possible.

Phosphate chain is held by all the three conserved amino acids of the P-Ioop

In view of the foregoing implication of A TP­induced changes in the polypeptide conformation, it was of interest to see portraits of the P-loop with bound nucleotides. Fig. 3 shows P-Ioop of the six subunits of F1-ATPase. The chains A, B, C and F have AMPPNP-Mg bound. The chain 0 has ADP-Mg bound. The chain E is nucleotide-free. It is interesting to note that the P-Ioop has only few contacts with the nucleotides, like a sickle pl aced on an inverted hammer. There are however some minor differences in interactions between the two types of subunits,

which must indirectly contribute to their distinctive roles.

ATP binds to each of the ~-subunits with equal low affinity and with hardly any hydrolysis. In presence of Mg2+, its binding cooperatively increases by 5-orders of magnitude accompanied by high catalysis 13. Interactions of its phosphate chain are coincidentally limited to the three conserved amino acids of the P­loop (GlyI61, Lys162 and Thrl63).

There are three interactions wit the phosphate chain in the catalytic ~-subunits . Some of these interactions between phosphate-O and the concerned atoms of the amino acids are shown in Table 3. The major one is through Mg2+, which coordinates with Thrl63-0Y of the P-Ioop on the one side and y­phosphate-O of A TP on the other. The other two significant interactions are the hydrogen bonds between LysI62-N~ and a terminal phosphate-O, and between Gly 161-amide-N and a ~-phosphate-O. The first two interactions with Thr 176 and K 175 occu r

RAMASARMA & RAMAKR[SHNAN : WALKER SEQUENCE [N FI -ATPase ACT[ON 11

Table 3 - Interaction of the nucleotides through hydrogen bonds and Mg co-ordination

[The di stances and the angles of interact ing bonds of nucleotides (ATP in chains A. B. C and F. and ADP in chain D) are shown. Where marked * Ol ~ instead of OIY in chain D. Those with weak interaction based on distance are shown in box]

No. Parameter Chain A Chain B Chain C Chain D Chain F

Dista1lces ( A ) 0 2Y----Mg 2.[5 2.07 2.[0 2.20

2 OIY (P) .... N~ IKI75(a). K I62(P)] ~ 13.731 3.32 *2.73 2.72

3 02~ (P)----Mg 2.23 2.30 2.17 2.29 2.44

4 OI~(P) ... . N [G[74(a),GI6I(P)] 3. 15 2.89 ~ 2.88 3.04

5 Olrl (P) . ... N [T[73(a),VI60(P)] 2.67 2.91 13 .31l1 13.321 IU9 6 OY [T [75(a),T 162(P)]----Mg 2.3 [ 2. 10 2.20 2.25 2.23

7 OJa (pa_O_ P~) .. .. N [G[74(a),G I61(P)] 2.9 1 2.85 2.67 13.271 ~ 8 N6 (adenine) .. .. O~ (Q430) 2.79 2.91 2.98 absent absen t

Angles ( 0 )

la 0 2Y (P)---- Mg----Ol (P)

2a OIY (P) .... N~ (K) -CE[KI75(a), K I629P»]

3a [l •

O2 (P)----Mg----OY [T 176( a),T 163(P)]

4a Ol ~ (P) .... N [G 174(a),G [61 (P)]- H(G)

5a Ol~ (P) ... . N [T[73(a),V [60(P)] - H(T/v)

6a OY [TI75(a).T [62(P)]----Mg----02 (pY)

7a 0 3a .... N[G 174(a),G 16 1(P)] - H(G)

8a C6- N6 (adenine) . . .. O~ (Q430)

also in a-subunits (chains A, B and C), but the third one with Gly 176 is rather weak. Interaction of the backbone with ATP (or ADP) thus appears to be

meaningful only in the ~-subunits. We consider thi s the single most important difference between the two subunits that marks them for separate actions.

More definition of nucleotide-backbone interaction specific to catalytic ~-subuni ts

It is to be noted that the tight site with high affinity to bind triphosphate could be observed only because of the use of high concentration of the non-hydro­lyzable analogue, AMPPNP, during crystallization). Thus this can be considered a locked, energized transition state. ATP is held through two oxygen atoms of its terminal phosphate by a hydrogen bond (2 .72 A) with Nt; (Lys162) , and by coordination (2.20 A) with Mg2+, also coordinated (2.23 A) with OY (TI63) at the other end. ATP exists in the P- loop fl eetingly . It appears that at any given time ADP occupies two sites and the third remains nucleotide free. Arriva l of ATP in thi s empty site disp laces the bound ADP in one of the two sites, and is itself rapidly hydrolyzed to ADP and Pi.

82 72 73 77

86 96 130 * I I [ Il[

83 90 90 90 83

17 37 32 27 10

20 35 37 3 1 40

167 159 164 160

35 40 38 28 43

107 113 120

The hydrogen bond between ~-phosphate-O and amide-N of G ly 161 selectively in the catalytic ~­subunits is a remarkable feature . Notice, this provides a direct interaction of the incoming A TP molecule with the polypeptide backbone. These atoms are located at an appropriate distance (3.04 A) for a

hydrogen bond . Such a hydrogen bond with ~­phosphate, combined with its Mg2+ coordination, securely holds the ADP portion of the molecule and virtually enables its bridge oxygen to undertake exchange of the third phosphate. This is unusual and deserves greater attention. It directs the spotlight on a backbone peptide unit, hitherto confined to secondary structure of the protein, for a share in action at the active site bes ides the favoured side chain res idues.

Hydrogen-bond network and other interactions in holding nucleotides

Mg2+ coordination bridging Thr-OY and a phosphate-O is the major force in holding A TP 111

both a- and ~-subunits (see Fig 3) . But the support provided by hydrogen bonding differs between the two subunits (Table 3). The corresponding P-Ioop g lycine (GlyI74) is in the same flipped state in the

12 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 39, FEBRUARY 2002

A B (

156

D E F

Fig. 3-- The P-Ioop bound with ATP or ADP. [The residues 169-176 of chains A-C and 156-\63 of chains D-F are arranged right to left. The peptide is sho'wn in the ball-and-stick representation. The nucleotide is in the stick representation . The chains A, B, C and F have bound ATP, chain 0 has bound ADP and chain E has no bound nucleotide. Also shown are the magnesium atom (Mg). and its dual coordination linkages to the peptide Thri63-0 and nucleotide phosphate-O. The side chains of the conserved residues, GKT, along with neighbours of G (T in chains A-C and V in chains D-F) are shown.]

three a-subunits (chains A, B and C), as in the nucleotide-bound ~-subunits (chains D and F). But its amide-N forms rather weak, bifurcated hydrogen bonds with an oxygen each from ~- and a-phosphates. The distance between the corresponding lysine-Ns (Lys 175) and the y-phosphate-O progressively decreases in A, Band C chains and comes close to having a hydrogen bond only in chain C in a­subunits. Concomitantly ~-phosphate-O and amide-N of Thrl73 , present in hydrogen bonding distance in A- and B-chains, progressively move away from each other in chain C (see Table 3).

The adenosine portion of a-subunits, chains A, B and C, is firmly held by hydrogen bonds. The amide­N6 of ATP forms a hydrogen bond with carbonyl-di of Gln430 (Fig. 4). There are hydrogen bonds also between the ribosyl-OR and ~-Arg372, and between adenine-N3 and ~-Tyr368, both amino acid residues chosen, strangely, from ~-subunits (not shown). On

the other hand, the adenine portion in ~-subunits is held in a hydrophobic pocket with little interactive specificity. This gross difference in forces within the protein that hold the adenosine portion of nucleotides may have significant effects on their specificity and action3. It offers an explanation why other nucleotides can replace ATP held (loosely?) in non-specific hydrophobic pocket in the ~-subunits, whereas multiplicity of binding forces makes ATP specific in a-subunits 14. For the same reason exchange and turnover of A TP in a-subunits may be absent.

Discussion

The P-Ioop, the site of action of ATP molecule, seems to have received less attention in the wake of excitement of the rotation of y-subunit within the stator made up of a3~3-subunits making F I-ATPase a tiny molecular motor lS

. This amazing, simple struc­tural design with so few amino acids is employed by a

RAMASARMA & RAMAKRISHNAN: WALKER SEQUENCE IN F,-ATPase ACTION 13

2

\ 8

~ A

B c

2

F D

Fig. 4---Interactions of the nucleotides with amino acid residues. [Portions of nucleotides are separately shown here, in stick representation, retaining only the interacting N, 0 atoms or the peptide units but omitting the peptide portion, for chains A-D and F. Orientations chosen are different, and these highlight the interactions of the phosphate chain with the conserved amino acids (GKT) of the P-Ioop. Coordination of Mg to Thr-OY and to phosphate-O of nucleotides (solid lines, 1,3,6), and hydrogen bonds (dotted lines, 2,4,5,7,8) between the Lys-N~ with terminal phosphate-O in all chains, and between the Gly-amide-N and ~-phosphate-O in chains D and F are shown. Notice adenosine portions of ATP have hydrogen bond (8) interactions in structural chains A, B and C, but not in catalytic chains D and F. The numbers on the lines indicate the interactions as in Table 3.]

variety of NTP-dependent reactions. A peptide unit in it flips on removal of the nucleotide as revealed by the crystal structure data of Walker's group3. This obser­vation is unique, yet remained unnoticed so far. Actually it is an excellent example of a bridge between the substrate and the backbone, and links the chemical reaction and the structural change.

Association with a ~-sheet on the N-side and an extendable a-helix on the C-side is also an invariant feature of the P-Ioop. In our opinion the whole of this sheet-loop-helix structure becomes 'alive' in action during energy transfer associated with NTP.

Simple switching between cis-trans isomers of its covalently bound retinal 16 forms the basis of transfer

14 INDIAN J. BIOCHEM. BIOPHYS .. VOL. 39. FEBRUARY 2002

of light energy in rhodopsin protein. Rotation in opposite direction of two adjacent bonds realized by a "very general externally applied strai n" causes this fas t photoi somerization accsirdi ng to an ab initio molecul ar dynamics study l7 .It is not unreasonable to expect that such structural changes constitute the flip of peptide unit in the P-loop. Imagine the value of such rotation of-a backbone peptide bond building up 'torsional energy' as tb,(f. link between energy forms. . '

Do not ignore the sirhple glycine just because it has no side chain . Absence of side-chains in two or more glycine units in thi s loop may offer an advantage' for movement of charged' phosphates in the space · thus created. Positioning of conserved Gly 161 is of far greater significance ' than thi s. The peptide unit between Val160 and Gly 161 flips its orientation on binding of ATP to the P-loop. Although information on the disposition of the peptide units and the interactions of nucleotides with the P-Ioop is available all the time in POB files, only this analysis based on the Ramachandran angles highlighted the flip . More importantly, the flip enables amide-N of Gly161 to form a hydrogen bond with ~-phosphate-O . Such a hydrogen bond also occurs between glycine-amide-N in the Walker sequence and· the nucleotide-phosphate­o in three 'other example~~ of transducin-a I8

, elon­gation factor-Tu 19 and a myosin motor domain2o

.

Hydrolysis of terminal phosphate of a nucleotide occurs in all these. Simplistically put, flip a peptide unit to break a P-O bond!

Asymmetry in the ~-subunits is known form the studies on binding affinities of Senior and colleagues21

. The nucleotide-free site one with highest affinity performs catalysis and this was demonstrated indirectly by using mutants and binding studies with the inhibitor, fluroaluminate that takes the position of Pi along with Mg-ADP. There is a view that all the three sites have bound nucleot ides in catalytic ' transition state' (see ref. 22 for an overview). Representing a post-hydrolysis and pre-product release state, this is possible after A TP is bound and before release of one of the two AOP, but is hard to identify, as the reactions are rapid. Ian Menz and coworkers23 obtained recently a structu re with nucleotide bound in all three sites by using fluroal uminate and Mg-AOP. They fo und that Gly 159-amide-N forms hydrogen bond with ~­phosphate-O, instead of Gly 161. This would not require a flip of its peptide uni t. By omitting Mg and thereby curtai ling hydrolysis , Mario Amzel and

colleagues24 obtained previously a "three nucleotide structure". In this structure the flip occurred at Gly 161 but its amide-N fo rmed hydrogen bond with a­phosphate-O, instead of ~-position . Additionally the peptide unit between Gly161-Lys 162 flipped and its carbon'yf-O formed a weak, bi furca[ed hydrogen bond with ~-phosphate-O . The above structures represent inactive, or more precisely "stop action", states.

. Nevertheless they point to the potential of peptide units forming part of the physic~-chemical changes.

The story of P-loop does not appear to be a copyright of nucleotide phosphates. Indeed the predictive dogma seems to have restricted search for this motif to nucleotide-binding proteins. And it proves right always. Who would look for it in superoxide dismutase or cytochrome peroxidase? And they have it. The Walker sequence is not exclusive for proteins dealing with nucleotide phosphates. But the sickle type P-loop is , and also it's conserved GKS for nucleotide binding.

In the foregoing definition and evaluation of the P­loop interactions, we took liberty to visualize and sketch the events occurring in, ,ATP hydrolysis. It represents a limited view, and '1lt' best promotes the value of the Walker sequence, particularly it's conserved residues, from the data given by Abrahams et at.3

, Indeed our pl acing so much of importance on the peptide flip found just in one structure may look speculative. If it is not found in the perception gained now, we will have to ii1Vent it as it offers a good model to magnify a simple chemical reaction through a lever-like twist of the backbone peptide chain into a 'global' mechanical change in a protein. Consequent to ATP hydrolysis, the major structural changes in the vicinity of the P-loop are the loss of ~-sheet on the N­terminus side and gain of sqme hydrogen bonds for an extra turn of a -helix on the C-terminus side (B-helix, 159-178)3. This will fit well with the proposal that energy exchanges may take place through reversible fo rmation of hydrogen bonds in proteins that can act as generators, carriers and utilizers2

, . The relationship of break of the P-O bond of ATP, the flip of the peptide, the twist of the backbone, and the change in the hydrogen bonds at a distance makes a fascinating study.

Large amount of structural and biochemical data of many previous authors was used in the present study. Particularly the first crystal struGture3 had greatly influenced our evaluation . This article is an example how systematic application of structural analys is

RAMASARMA & RAMAKRISHNAN: WALKER SEQUENCE IN FI-ATPase ACTION 15

using the Ramchandran angles and side chain torsion angles of peptide fragments existing in different states can bring forth new findings and explanations of a phenomenon from the recorded data. Hopefully peptide flip in the P-Ioop will be a driving force in understanding biological hydrolysis and synthesis of ATP.

Acknowledgement CR acknowledges financial assistance for this

project from the Council of Scientific & Industrial Research, New Delhi. TR is a senior scientist of Indian National Science Academy, New Delhi. Part of this article is based on a presentation at the meeting of TRendys in Biochemistry at the University of Pune (July 30, 2001), and TR thanks the conveners, Prof. V. Sitaramam, Dr. G.c. Mishra and Dr. V. Sheorain for giving this opportunity. This article is dedicated to Prof. G. N. Ramachandran (passed away on April 7, 2001) whose work on the torsion angles and the map forms the core of this investigation.

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