THE JOURNAL 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

OF BIOLOGICAL CHEMISTRY Vol. 267, No. 31, Issue of November 5, pp. 22328-22335,1992 Printed in U.S.A.

Glutaraldehyde Cross-links Lys-492 and Arg-678 at the Active Site of Sarcoplasmic Reticulum Ca2’-ATPase*

(Received for publication, June 10, 1992)

David B. McIntosh From the Medical Research Council Biomembrane Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa

It has been shown previously that glutaraldehyde cross-links the Caz+-ATPase of sarcoplasmic reticulum intramolecularly at the active site, involving residues participating in nucleotide binding and the conforma- tional change that results in Caz+ release to the vesicle lumen and formation of ADP-insensitive E,-P (Ross, D. C., Davidson, G . A., and McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621). This study shows that 10 nmol of [14C]glutaraldehyde/mg of protein attached irreversibly to the ATPase under conditions optimal for formation of the intramolecular cross-link. Half of this amount (i.e. 1 mol/mol ATPase) was inhibited by nucleotide binding. Thermolysin digestion of deriva- tized vesicles released two nucleotide-sensitive 14C- labeled species, which were isolated and identified as

FSRDR*S and FSRDR*S FA* FA*VEPS

where the missing residues are Lys-492 and Arg-678. The majority of the 14C label was released in the sixth cycle of both Edman degradations, confirming the cross-link position. Lys-492 and Arg-670 are evi- dently close together in the active site, but their dis- tance apart in the linear sequence suggests that they may arise from separate domains, which together con- stitute an ATP binding cleft. Residues in both regions, and Lys-492 in particular (McIntosh, D. B., Woolley, D. G., and Berman, M. C. (1992) J. Biol. Chem. 267, 5301-5309), have been derivatized by nucleotide- based affinity probes. Mutations of both of these resi- dues in some of the bacterial P-type ATPases suggest that they do not play an essential catalytic role, and the inability of the cross-linked ATPase to form Ez-P and to release Ca2+ to the lumen is probably because an essential tertiary structural movement at the active site is blocked.

The 110,000-dalton Ca2+-ATPase of skeletal muscle sarco- plasmic reticulum (SR)’ belongs to a family of ATP-depend-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SR, sarcoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; MOPS, 3-(N-morpholino)propanesulfonic acid; AMP-PCP, adenyl-5”yl methylenediphosphonate; TNP-~Ns-AMP, 2’,3’-0-(2,4,6-trinitrophenyl)-8-azido-adenosine monophosphate; TNP-8Ni,-ATP, 2’,3’-0-(2,4,6-trinitrophenyl)-8-azido-adenosine tri- phosphate; E(110), native Ca2+-ATPase migrating on SDS-PAGE with the correct molecular mass of approximately 110,000 daltons; E(125), intramolecularly cross-linked Ca2+-ATPase migrating on SDS-PAGE with an apparent molecular mass of 125,000 daltons; PTH, phenylthiohydantoin.

glutaraldehyde Reactiwty to

HoH ’ El- p ......................... Ca reactwe

most

HOH J

..........................

SCHEME 1

ent cation pumps in which phosphorylation of an aspartyl residue at the active site is an essential step in the transport mechanism (for recent reviews see Refs. 1-3). We have pre- viously described a novel reaction of glutaraldehyde and other aldehyde based cross-linkers with the Ca2’-ATPase that re- sults in the formation of a stable intramolecular cross-link, at a site blocked by ATP binding to the active site (4-7). The cross-link alters the mobility of the protein during sodium dodecyl sulfate polyacrylamide gel electrophoresis, and this has permitted quantitation of the reaction. Scheme I shows the reactivity of the principal catalytic intermediates to glu- taraldehyde and the functional consequences of introducing the cross-link (6, 7). Ca2+ and/or M$+ binding has no effect on the cross-link reaction at alkaline pH, but phosphorylation to the ADP-sensitive El-P(2Ca) catalytic intermediate with occluded Ca2+ ions enhances the rate 3-4-fold, whereas phos- phorylation to the ADP-insensitive E2-P intermediate blocks cross-linkage. We interpreted the three cross-link-sensitive conformational states of the active site, which accompany Ca2+ binding, occlusion, and release to the lumen, as reflecting two sequential hinge-bending movements leading to closure of the site (6).

The reactions carried out by the cross-linked enzyme are indicated by bold arrows in Scheme I. The cross-link decreases the affinity of the ATPase for nucleotides by more than 2 orders of magnitude, which is consistent with nucleotide binding inhibiting formation of the cross-link. It slows phos- phoryl transfer from ATP or to ADP by 3 orders of magnitude, but phosphorylation by small substrates such as acetyl phos- phate is not affected or is possibly accelerated (step 2). High levels of ADP-sensitive El-P(2Ca) are formed, even with ATP, due mainly to inhibition of the next step involving a conformational change of the protein (step 3). The catalytic cycle of the cross-linked ATPase follows an uncoupled reac-

22328

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Glutaraldehyde Cross-link Site of SR Ca2+-ATPase 22329

tion of El-P(2Ca) with water and Ca2+ release to the exterior (step 7). Phosphorylation from Pi is blocked (step 5 , reverse direction). The inability to form E2-P is consistent with the finding that this catalytic intermediate cannot be cross- linked.

In this study, we have used [14C]glutaraldehyde to identify the cross-link site of the Ca2+-ATPase. We show that glutar- aldehyde reacts fairly specifically at a unique cross-link site connecting residues Lys-492 and Arg-678. Lys-492 has been recently shown by us to be photoaffinity labeled by TNP- 8N3-AMP and -ATP (8,9). Arg-678 has not yet been affinity labeled, but 2,6-butanediol modifies an active site residue (lo), which may be this one, and we have shown previously that phenyl glyoxal blocks the cross-link ( 5 ) . Neighboring Lys-684 is derivatized by pyridoxal ATP in the presence of Ca2+ (11) and has been shown to be essential by site-directed mutagenesis (12). In the absence of Ca2+, pyridoxal ATP labels both Lys-492 and Lys-684 (13). Both regions, and the 3 residues in particular, can now be placed in close proximity at the catalytic site, and the effects of the cross-link can be rationalized in terms of their role in nucleotide binding and essential movements between them.

Despite the routine use of glutaraldehyde as a protein cross- linking reagent, this appears to be the first example of a stable glutaraldehyde cross-link involving the monomeric di-alde- hyde and not requiring borohydride reduction. A mechanism is proposed for this cross-link in which stabilization is achieved by formation of a resonating cyclic carbinolamine derivative. I t is possible that, at low concentrations of reagent, cross-linking of proximal lysyl and arginyl residues by alde- hyde-based cross-linkers may not be uncommon because of imine and alkyl-ol stabilization through reaction with the guanidinium group.

EXPERIMENTAL PROCEDURES

Materials-The sources of chemicals were as follows: AMP-PCP, hydrazine, and glutaraldehyde (grade I, 25% solution), Sigma; ther- molysin, Boehringer Mannheim; [1,5-'4C]glutaric acid, ICN Radi- ochemicals; oxalyl chloride, bis(triphenylphosphine)copper(I) tetra- hydroborate, and triphenylphosphine, Aldrich.

SR vesicles were prepared by the method of Champeil et al. (14), with modifications as described previously (6). Protein determination was based on the Lowry method and was performed as described before (9).

[14C/Glutaraldehyde Synthesis-The synthesis of radiolabeled glu- taraldehyde was adapted for small quantities from the method of Barna and Robinson (15). A freshly prepared mixture of oxalyl chloride and triethylamine (320:1, v/v, 85 pl) was added to glutaric acid (200 pmol, made up of 7.2 pmol (according to manufacturer) of [1,5-'4C]glutaric acid (250 pCi) + 192.8 pmol of unlabeled glutaric acid). When effervescence ceased the mixture was warmed briefly. Excess oxalyl chloride was then removed under a stream of nitrogen. The remaining liquid was diluted with acetone (0.2 ml) and added slowly (over 10 min) to a vigorously stirred slurry of bis(tri- phenylphosphine)-copper(1) tetrahydroborate (0.26 g, 0.44 mmol) and triphenylphosphine (0.23 g, 0.88 mmol) also in acetone (0.8 ml). The reaction was continued for 2 h. The suspension was diluted with water and then filtered. The filtrate was centrifuged at 10,000 rpm for 20 min to obtain a clear supernatant. The glutaraldehyde solution was purified by passage first through a column (0.7 X 9 cm) of CM Sephadex C-50 and then a column (0.7 X 9 cm) of DEAE-Sephadex A-50, both equilibrated with water. It was stored at -20 "C as a 6 mM solution in water and found to be stable, in terms of the rate of formation of the intramolecular ATPase cross-link and purity as judged by thin layer chromatography and HPLC, for over 3 years. The specific activity was 1800 cpm/nmol, assuming all the glutaral- dehyde was present as the free monomeric form, and the yield 4476, both based on gravimetric analysis of the 2,4-ditritrophenylhydrazone derivative. The prepared ['4C]glutaraldehyde and the commercial unlabeled product (see above) formed the intramolecular cross-link at similar rates. Both chromatographed as a single species on thin layer chromatography (silica gel; solvent: chloroform/methanol

(19:1), visualized by iodine vapor, RF = 0.45). The radioactivity of the [14C]glutaraldehyde, in addition, was largely (>95%) associated with the single visible spot seen by thin layer chromatography (plate scanned by Berthold LB2723 proportional gas flow counter).

Cross-linking Reaction-The standard cross-linking medium was 30 mM MOPS/tetramethyl ammonium hydroxide, pH 8.1, 0.2 M sucrose, 10 mM KCI, 0.05 mM CaC12, 0.142 mM glutaraldehyde, and 0.5 mg of SR protein/ml. The reaction was carried out a t 25 "C for 1 h unless indicated otherwise. The method of stopping the reaction depended on the purpose of the experiment. For quantitation of the reaction of ['4C]glutaraldehyde with SR vesicles, the reaction was stopped by filtering aliquots (0.25 mg of protein; Millipore filters, type HAWP 002500) and washing briefly with 50 mM potassium phosphate, p H 7.0. The filters were assayed for radioactivity in 10 ml of Insta-Gel I1 (Packard). If the samples were to be subjected to SDS- PAGE, it was stopped by diluting aliquots 2-fold with 200 mM Tris/ HCl, pH 6.8, 2% SDS, 8 M urea, 500 mM 2-mercaptoethanol, and containing a few dissolved grains of bromophenol blue. If the samples were to be digested, the reaction was stopped with a 4-fold molar excess of hydrazine over glutaraldehyde.

Thermolysin Digestion, HPLC, and Peptide Sequencing-SR vesi- cles (2 or 10 mg of protein, as indicated in the figures) were cross- linked for 1 h and the reaction quenched as described above. Volumes of 3 ml of the suspension were each passed through a Sephadex G-25 M PD-10 column (Pharmacia) pre-equilibrated with 25 mM NH4HCOs, pH 7.5. Fractions (0.5 ml) were collected, and peak frac- tions were pooled (3 ml from each column). CaC12 was added (final concentration 5 mM), and the vesicles were digested with thermolysin (4% (w/w) of SR protein) a t 40 "C for 1 h. The digestion mixture was passed through a C18 Sep-Pak cartridge (Waters), and the soluble peptides retained on the column were eluted with 1 ml of 10 mM potassium phosphate, pH 6.5, 60% (v/v) acetonitrile. The concentra- tion of acetonitrile was lowered by blowing a stream of nitrogen over the surface, and the sample was filtered through a Millipore filter (type HV) prior to HPLC. The first HPLC was performed with a Vydac C4 reverse phase column (0.46 X 25 cm) equilibrated with solvent A (10 mM potassium phosphate, pH 6.5) and connected to a Beckman System Gold HPLC. The column was developed with solvent B (10 mM potassium phosphate, pH 6.5,60% (v/v) acetonitrile a t a flow rate of 1.5 ml/min with the following gradient: 0% B for 10 min, 0-20% B (linear) over 40 min, 20-100% B (linear) over 5 or 25 min (see relevant figure for which each is applicable), 100% B for 5 min, 100-0% B over 5 min. The elution was monitored a t 224 nm, and fractions (0.75 ml) were collected a t 30-s intervals and assayed for radioactivity. In the case of the bulk sample (10 mg of protein), the fractions indicated in the appropriate figure were pooled, and the acetonitrile concentration lowered by blowing with nitrogen as above, reinjected in multiple aliquots onto a Vydac CIS reverse phase column pre-equilibrated with solvent A, and developed at a flow rate of 1.5 ml/min with solvent B with the following gradient: 0% B for 5 min, 0-13% B (linear) over 5 min, 13-30% B (linear) over 45 min, 30- 100% B (linear) over 5 min, 100% B for 5 min, and 100-0% B over 5 min. The elution was again monitored at 224 nm and peak fractions collected and assayed for radioactivity. The two fractions containing the majority of the radioactivity (indicated in the appropriate figure) were subjected to a stream of nitrogen as above, were filtered, and were separately reinjected onto a Vydac C18 reverse phase column preequilibrated with solvent C (0.1% (v/v) trifluoroacetic acid). The column was developed with solvent D (0.1% (v/v) trifluoroacetic acid/ 60% (v/v) acetonitrile) at a flow rate of 1 ml/min with the following gradient: 0% D for 5 min, 0-100% D (linear) over 45 min, 100% D for 5 min, and 100-0% D over 5 min. The elutions were monitored a t 224 nm and peak fractions collected and assayed for radioactivity. The two fractions from each chromatography containing radioactivity (see appropriate figure) were pooled and freeze-concentrated under vacuum to approximately 30 pl. Peptide sequence analysis was per- formed by W. Brandt (Department of Biochemistry, University of Cape Town, Rondebosch, Cape Town 7700, South Africa) using standard procedures. Approximately one-third of the sample follow- ing each Edman degradation cycle was converted to the phenylthio- hydantoin derivative, and the rest was assayed for radioactivity. Amino acid analysis was performed by G. Rodrigues, of the same department, by acid hydrolysis, conversion to the phenylthiocarbamyl derivatives, and identification of the latter by standard HPLC.

Gel Electrophoresis and Quantitation-SDS-PAGE was performed according to Laemmli with 7.5% acrylamide and stained with Coo- massie Blue. The relevant bands were excised and dissolved in l ml of Hz02 by heating a t 70 "C overnight. Insta-Gel I1 (10 ml) was added,

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22330 Glutaraldehyde Cross-link Site of SR Ca2+-ATPase

and the samples were stored for 3 days in the dark and then assayed for radioactivity. The amounts of ATPase in the E(110) and E(125) bands were determined from the total amount of protein applied to the gel and the percentage of protein in each band obtained from gel scans.

ATPase Actioity-Cat+-dependent ATPase activity was carried out at 25 "C using an enzyme-linked assay with 1 mM ATP (6). Aliquots of SR vesicles taken during the standard cross-linking reaction were diluted a t least 100-fold into ice-cold assay medium and the activity measured within 1 h.

RESULTS

Reaction of [I4C]glutaraldehyde with SR vesicles is shown in Fig. 1. The conditions chosen have previously been shown to be optimum for formation of the intramolecular cross-link (6). The assay was performed by filtering aliquots, washing the filters briefly, and assaying the filters for radioactivity. The results show that 40-50 nmol of glutaraldehyde/mg of protein associate with the vesicles in 2 h. This is equivalent t o approximately 10 mol/mol active ATPase; maximum phos- phoenzyme levels are 4-5 nmol/mg of protein in these prep- arations (6, 9). If the reaction is carried out in the presence of 0.1 mM AMP-PCP, the extent of labeling is 5 nmol/mg of protein less after 1-2 h, suggesting that 1 mol of sites/mol active ATPase is protected against reaction with glutaralde- hyde.

There are several ways in which glutaraldehyde could be associated with the vesicles as measured by such a filtration assay. These include irreversible reaction with protein or lipid components, or, if reversible, not fully reversed during the washing period, intercalation into the lipid bilayer and only partial dissociation during the wash, and luminally located glutaraldehyde. We have shown previously that the cross-link at the active site can be quantitated by SDS-PAGE because of the retarded mobility of the cross-linked compared with the uncross-linked protein (4). The ability to separate the two species provides an unusual opportunity to quantitate the chemical modification separately for each species from the gel bands and also of determining the amount of glutaraldehyde associated irreversibly with the ATPase. The relevant portion of the gels following reaction of SR vesicles with [I4C]glutar- aldehyde and the results of the quantitation are shown in Fig. 2 ( A and B) . Note that the amount of radioactivity associated with each band is expressed per mg of protein in that band. The uncross-linked (E(110), in the presence of 0.1 mM AMP- PCP), and cross-linked protein (E(125)) exhibited the same linear incorporation of glutaraldehyde with time to the extent of 7.5 and 12.5 nmol/mg of protein, respectively. Significantly, the E(125) species had an extra 5 nmol of glutaraldehyde/mg of protein associated with it, which must be due to the stable intramolecular cross-link at the active site. Thus, there are approximately 2 mol of glutaraldehyde/mol ATPase associ-

50 I I

Tim? (mh)

FIG. 1. Reaction of glutaraldehyde with SR vesicles. SR vesicles were incubated for the times shown in standard cross-linking medium without (solid circles) or with (open circles) 0.1 mM AMP- PCP and then filtered. The filters were washed briefly and then assayed for radioactivity.

(+AMPPCP

E(125)-

- f 6v/

3 3 \E( I 10) + AMPPCP 0

20 " t c . -1 o 20 40 60 80 loo 120

FIG. 2. Irreversible reaction of glutaraldehyde with Ca2+- ATPase and effect on ATPase activity. SR vesicles were reacted with glutaraldehyde under standard conditions without or with 0.1 mM AMP-PCP, and aliquots were taken at the times indicated for SDS-PAGE and ATPase activity measurements. In A, the section of the Coomassie Blue-stained gel containing the uncross-linked (E(110)) and cross-linked (E(125)) ATPase is shown. In B, the time dependence of glutaraldehyde incorporation into the E(125) band in A(i) and the E(110) band in A(ii) is shown. In C, the time dependence of inactivation of Ca"-dependent ATPase activity in the absence of glutaraldehyde (open squares) and in the presence of glutaraldehyde and absence (open diamonds) or presence (open triangles) of 0.1 mM AMP-PCP is shown. Also included is the percentage of E(110) remaining in A(i) (solid diamonds) and in Afii) (solid triangles) ob- tained from gel scans (data expressed as (E(110) at each timelE(110) a t time 0.1 min) X 100). The lines have been drawn through the ATPase activity data points.

iime (min)

ated irreversibly with the ATPase, 1 mol of which is at the specific cross-link site and the other, according to the linear rate of incorporation, a t mainly nonspecific sites.

The effect of the glutaraldehyde reaction on Ca*+-depend- ent ATPase activity is shown in Fig. 2C. Both in the absence and in the presence of 0.1 mM AMP-PCP, ATPase activity declined in parallel with the decrease in the E(110) species. This correlation suggests that reaction of glutaraldehyde a t sites other than the active site and not resulting in intermo- lecular cross-linking has no effect on activity, which, in turn, suggests that any such reaction is nonspecific, in agreement with the linear kinetics of glutaraldehyde incorporation into the E( 110) species.

HPLC analysis of the soluble peptides produced by ther- molysin digestion of the derivatized ATPase confirmed the uniqueness of the intramolecular cross-link site (Fig. 3). The digest contained two main peaks of radioactivity eluting a t 47 and 56 min. The earlier eluting peak was absent when the reaction was performed in the presence of AMP-PCP, and

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Glutaraldehyde Cross-link Site of SR Ca2+-ATPase 22331

- c 800

600 - AMPPCP

200

[L ,_;::

+ AMPPCP

400 2

0 10 20 30 40 50 KO

Retention time ( rn tn )

FIG. 3. HPLC of soluble thermolysin digestion products fol- lowing reaction of SR vesicles with glutaraldehyde. SR vesicles (2 mg of protein) were reacted with glutaraldehyde under standard conditions without (top) or with (bottom) 0.1 mM AMP-PCP for 1 h. The suspensions were then passed through a Sephadex PD-10 column to remove most of the unreacted glutaraldehyde and change the medium to 25 mM NH4HC03, pH 7.5. Ca2+ and thermolysin were added and the digestion continued for 30 min at 40 "C. The suspension was passed through a CIS Sep-Pak cartridge, and the soluble peptides retained on the cartridge were eluted and subjected to HPLC on a C, column, with solvent A as 10 mM Pi, and solvent B 10 mM Pi/60% acetonitrile. The elution was monitored at 224 nm, and fractions were collected at 30-s intervals and assayed for radioactivity.

this strongly suggests its association with the specific intra- molecular cross-link. The later eluting peak is largely a con- sequence of the steep gradient used in this part of the chro- matography, as will be shown below.

The HPLC profiles following bulk labeling and purification of the species contained in the nucleotide-sensitive peak are shown in Fig. 4. The first C4 HPLC was performed with a similar gradient to that shown in Fig. 3, except that in the final step the gradient was shallower and over a longer time. The profiles obtained are shown in Fig. 4 (top frame) and are similar to the analytical profiles except that the peak of radioactivity eluting at 57 min has been split into two as a result of the shallower gradient and becomes less significant. The nucleotide-sensitive peak was easily identified as that at 46 min. Rechromatography of the pooled fractions making up this peak on a CIS column with a shallow gradient of phos- phate/acetonitrile, pH 6.5, resulted in two poorly separated peaks containing virtually all the radioactivity (Fig. 4, second frame from top). The earlier eluting fraction yielded a single radioactive peak eluting at 24 min on rechromatography in trifluoroacetic acid/acetonitrile solvent system (Fig. 4, third frame from top). The later eluting fraction contained a mix- ture of the first species (elution time 24 min) and another species eluting at 26 min (Fig. 4, bottom frame). On the basis of radioactivity, the proportion of the two species is approxi- mately 6:l. The yields at each step are shown in Table I. They are similar to those we obtained previously in the isolation of a peptide fragment derivatized with TNP-8N3-AMP and

Representative sequencing results of the two species are shown in Table 11. Following the Edman degradation, one- third of the sample was converted to the phenylthiohydantoin derivative and the amino acid identified by HPLC and the remainder was assayed for radioactivity. The majority of the radioactivity was recovered in the 6th cycle in both cases. The

-ATP (9).

1 c4 1 3000

, , llo.o " pH 6.5 2000 2

10 20 30 40 50 60 70 80

Retention time (rnin)

FIG. 4. Isolation of nucleotide-sensitive ["C]peptides by HPLC. Soluble peptides generated as in Fig. 3 following bulk labeling (10 mg of protein) and thermolysin digestion were subjected to C, HPLC, with solvent A as 10 mM Pi and solvent B 10 mM Pi/60% acetonitrile and the gradient shown (top frame). Note the gradient after 50 min is not as steep as that in Fig. 3. Fractions were collected at 30-s intervals and aliquots assayed for radioactivity. Fractions at 45-48 min were pooled and reinjected onto a C18 column with the same solvents as before with the gradient shown (second frame from top). Peak fractions were collected and aliquots assayed for radioac- tivity. Peaks eluting at 34 and 35 min were reinjected separately onto the same C I S column with solvent A as 0.1% trifluoroacetic acid and solvent B 0.1% trifluoroacetic/60% acetonitrile with the gradient shown (third and bottom frames, respectively). Peak fractions were collected and assayed for radioactivity. In all HPLC profiles peptides elutions were monitored at 224 nm.

sequences and cross-linked residues deduced from the anal- yses and scrutiny of the known amino acid sequence of the ATPase polypeptide (16) are shown in Table 111. In both cases the cross-linked residues are Lys-492 and Arg-678. Analysis of the second smaller fraction was complicated by a contam- inating peptide, but it was not difficult to determine the sequences with the help of the amino acid sequence of the protein and the realization that the cross-link was the same as in the more abundant species. The two cross-linked species are obviously the result of partial cleavage of the Arg-678/ Val-679 peptide bond by thermolysin. It is interesting to note that the contaminating peptide has a similar amino acid composition to the cross-linked peptides, which explains their copurification. The bulk derivatization, peptide isolation, and sequencing were repeated with another SR preparation with

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22332 Glutaraldehyde Cross-link Site of SR Ca'+-ATPase TABLE I

Bulk labeling and 14C-adduct recoveries

Step Description Labeling Recovery

Overall Stepwise

cpm nmol % % 1 Reaction" 224,180 88 100 100 2 Digestion and 131,740 52 59 59

Sep-Pak separation

3 HPLC Cdb 23,900 9.4 11 18 4 HPLC Cls(i)

Fraction 1 6,800 Fraction 2 5,280 2.1

5 HPLC Cdii) Fraction a (Combined) Fraction b 840 0.3

2.7 1 5.4 51

4'890 l.gl 2.6 47

Values were obtained from Fig. 2 for 10 mg of protein and refer to irreversible reaction with ATPase.

* Pooled fractions 45-48 min; see Fig. 4.

TABLE I1 Edman degradations of fractions a and b

Following PTH derivatization approximately one-third of the sam- ple was subjected to HPLC for PTH-amino acid (PTH-AA) identifi- cation and the remainder was assaved for radioactivitv.

Fraction a Cycle

Fraction b

PTH-AA (pmol) cprn PTH-AA (prnol) cprn

1 F (1675) 0 F (1000); I(239) 0 2 S (315); A (824) 0 A (574); S (174); 0

3 R (268) 0 R (176) 0 4 D (566) 0 S (210); D (244); 0

5 R (306) 0 L (431); E (280); 0

6 367 P (438) 80 7 s (153) 71 S (235) 24 8 25 V (140) 9 9 6 E (264) 12

10 0 T (66) 0 11 Not determined 0 12 Not determined 0

V (236)

V (180)

R (110)

TABLE 111 Deduced amino acid sequences and cross-link sites

Fraction a Fraction b

I V R S L P S V E T

a similar result, except that the yields of the two cross-linked peptides were about one-third and the contaminating peptide in the second fraction was absent.

An amino acid analysis was performed on fraction a to establish whether or not the cross-link was stable to acid hydrolysis, and the results are shown in Table IV. It is apparent that the cross-linked lysyl and arginyl residues were regenerated by acid hydrolysis and the mol residue/mol frag- ment confirms the sequencing results.

DISCUSSION

Identification of the residues cross-linked by glutaraldehyde as Lys-492 and Arg-678 brings together two regions in the tertiary structure of the SR Ca2+-ATPase that are widely separated in the linear sequence. Both regions (and Lys-492

TABLE IV Amino acid analysis of fraction a

Amino acid PTC-AA rnol/mol"

nmol

F 3.5 2 S 3.2 2 A 1.7 1 R 5.1 3 D 2.1 K

1 1.5 1

Values were obtained by dividing the amount of phenylthiocar- bamyl amino acid (PTC-AA) found by 1.7 nmol.

in particular) have been labeled by affinity probes of nucleo- tide binding sites (see below), and it is now certain that they together constitute the catalytic nucleotide binding site. The profound effects of the cross-link nucleotide binding, cataly- sis, and transport (6, 7) suggest the involvement of these two residues in substrate binding and essential tertiary structural changes at the active site.

Glutaraldehyde Reaction-Glutaraldehyde has been exten- sively used as a general cross-linking agent because of its ability to generate multiple stable cross-links without the need for borohydride reduction. The concentrated reagent consists of an equilibrium mixture of the linear di-aldehyde monomer, the cyclic hemiacetal monomer, polymers of the latter, and, at alkaline pH, unsaturated aldehyde polymers (17-20). The reactions of the concentrated reagent with pro- teins involve principally lysyl residues and are suggested to result in 1,3,4,5- pyridinium salts (19) and/or conjugated Schiff base linkages (17, 20). The cross-links are stable to acid hydrolysis. The postulated mechanisms dictate that a stable cross-link between two lysyl residues is constituted from a minimum of three glutaraldehyde monomers linked together. The cross-link in this study was obtained at lower concentrations of glutaraldehyde than used in most investi- gations, was unstable to acid hydrolysis, and the involvement of monomeric di-aldehyde and a lysyl and an arginyl residue point to another mechanism generating this stable cross-link.

A postulated structure of the cross-link and possible reac- tion scheme are shown in Fig. 5. In this mechanism, a Schiff base is formed with Lys-492 and a carbinolamine with Arg- 678. The carbonium ion produced by protonation of the imino nitrogen of the Schiff base is then subject to nucleophilic attack from one of the nitrogen atoms of the guanidinium grouping. Reaction with the nitrogen proximal to the hy- droxyl, as illustrated, results in a six-membered piperidine ring derivative or cyclic carbinolamine (iii) and is likely favored over the eight-membered alternatives (nitrogen atoms labeled 2 or 3). Cyclic carbinolamines are known to be unsta- ble and in equilibrium with the free aldehyde (Fig. 5 , iu; Ref. 21). However, in this case, the unsaturated nature of the guanidinium grouping may provide extra stabilization of the cyclic species through resonance forms involving the tertiary amino group. This stabilization is evidently not sufficient to resist acid hydrolysis. It is interesting that the reaction prod- uct of low concentrations of formaldehyde and the guanidi- nium group of arginine is a N,N-bis-(hydroxymethyl) guani- dinium derivative, also probably stabilized by the unsaturated nature of the guanidinium group (22). This structure was stable enough to be isolated but was hydrolyzed in acid. Structure iu, with the possibility of an extra cross-link via the regenerated aldehyde, was entertained as a possible reason for three sequences appearing in fraction b (Table 111) but was rejected when a second bulk purification failed to reveal the third peptide sequence.

The proposed mechanism places the amino and guanidinyl

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22333

i I I i i i FIG. 5. Proposed cross-link structure and mechanism.

i v

groups of the amino acid residues at the active site within a few angstroms of each other. Such proximity is also suggested from the finding that glyoxal (4), formaldehyde (4), and o- phthalaldehyde' form a stable intramolecular cross-link, which alters the mobility of the modified proteins in SDS- PAGE in the same way as the glutaraldehyde cross-link does. I t is also pertinent to the reaction mechanism that mono- aldehydes (for example acetaldehyde, glycolaldehyde, and benzaldehyde) or incorrectly spaced or positioned di-alde- hydes (for example m- andp-phthalaldehyde) do not form the cross-link.'

It remains to be seen whether the Lys-Arg cross-link is common in the reaction of low concentrations of glutaralde- hyde with proteins. The fast reaction with Lys-492 and Arg- 678 is undoubtedly due in part to the unusually low pK, of Lys-492 (pK, = 7.5) (8, 9). I t seems possible however that at low concentrations of glutaraldehyde, and therefore predom- inance of the monomeric species, it may be a favored reaction for producing a cross-link because of the unique stabilization afforded by the guanidinium group. The similar cross-links produced by other aldehyde-based cross-linkers, noted above, suggest that the reaction is not limited to glutaraldehyde.

Location and Conservation of Cross-linked Residues-Lys- 492 and especially Arg-678 lie in regions of the polypeptide where the conservation of amino acid residues is high. The region around Lys-492 has been compared with other AT- Pases in our previous study of labeling of this residue by TNP-8N3-AMP and -ATP (9) and will not be repeated here. Of interest, is that although Lys-492 is conserved in all animal and plant ATPases sequenced to date, it has apparently been replaced in bacterial ATPases. If alignments are correct (see also Ref. 23), it appears to be replaced with an arginyl residue in the K+-ATPase of Escherichia coli, and a glycyl residue in some other bacterial pumps. The equivalent residue of the pig gastric H+,K+-ATPase (Lys-497) has been derivatized with pyridoxal 5'-phosphate (24) that of the lamb kidney Na+,K+- ATPase (Lys-480) with pyridoxal 5"phosphate and pyridoxal ADP (25). 8-Azido-ATP labels a peptide fragment of the Na+,K+-ATPase in this region (26).

The amino acid residues in the region around Arg-678 in the Ca2+-ATPase are compared with several other examples of animal, plant, and bacterial ATPases in Fig. 6 (see also Ref. 23). The examples of the latter ATPases have been chosen to illustrate the variety of replacements of Arg-678, which include glutamyl, glutaminyl, and glycyl residues. The region shown is one of the most highly conserved in the protein, and it has been uncertain whether it forms a separate hinge domain or is an integral part of the catalytic nucleotide binding site (12, 27). I t is now clear from the present results that at least part of it constitutes the nucleotide binding site, consistent with affinity labeling of surrounding residues.

'' D. C. Ross and D. B. McIntosh, unpublished observations.

Close to Arg-678 is Lys-684, which, along with intervening Pro-681, is absolutely conserved in all ATPases sequenced so far. Interestingly, and in agreement with the identity of the cross-link sites, Lys-684 is derivatized with pyridoxal-ATP in the presence of Ca2+ (l l) , and both Lys-492 and Lys-684 are labeled in the absence of Ca2+ (13). Lys-684 is essential according to a site-directed mutagenesis study (12). A large section on the amino-terminal side of Arg-678 is present in the plant and animal ATPases and absent in the bacterial pumps (number of missing residues shown in parentheses). This is followed by another conserved region containing the conserved triplet Thr-Gly-Asp. This segment in the yeast plasma membrane H+-ATPase (Asp-560 to Lys-566) is labeled by 2-azido-ATP (28), and mutations in the triplet impaired transport and phosphorylation from both ATP and Pi (29). On the carboxyl-terminal side of Arg-678 the triplet is re- peated again in another highly conserved section, wherein Lys-712, labeled with 5'-(p-fluorosulfonyl)benzoyladenosine in the dog kidney Na+,K+-ATPase (Lys-725) (30), is found. Mutations in the triplet and neighboring residues either blocked phosphorylation from ATP and Pi or impaired trans- port by inhibiting the E1-P(2Ca) to E,-P transition (29).

In view of the absolute inhibitory effect of the cross-link on Ca'+ transport (7) and the probable proximity of the amino and guanidinium groups of the cross-linked residues, it is interesting to examine which pairs of residues occur in the sequences so far known. If the alignments are correct, the following pairs are found (N-terminal residue first): Lys/Arg (Ca2+-, Na+,K+, and H+-(gastric) ATPases); Lys/Gln, Lys/ Gly, and Lys/Glu (H+-ATPases, plants); Arg/Glu, Gly/Glu, and Gly/Gly (various bacterial ATPases). I t is surprising to note the Lys/Glu (H+-ATPase yeast) and Arg/Glu (K+-ATP- ase E. coli) pairs, which might be expected to form a salt cross-link. The dire functional consequences of the Lys-492 and Arg-678 glutaraldehyde cross-link suggest that the basic/ acidic pairs do not in fact interact. The substitutions suggest that the cross-linked residues are not involved directly in phosphoryl transfer but rather in substrate binding and the preferred replacement of either residue with Gly hints at these residues being involved in bending movements.

Structural and Functional Implications-The position of the cross-linked residues and other residues labeled in the Ca2+-ATPase and other related ATPases by nucleotide affin- ity probes is shown in Fig. 7. It is unclear how the targeted residues are topographically constituted into an active site, but the distances between Asp-351, Lys-492, and Arg-678 in the linear sequence suggest that they each belong to separate domains, which together constitute the active site. The region around Lys-492 has been previously segmented into three p- strands based on secondary structural predications and there may be more (9). This region could form one side of a cleft and the region around Arg-678 the other side, with the phos-

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22334

C a (SSR)

Ca(HPL)

N a (Tor)

H(Hgas)

H(Neur)

? (Ldon)

K ( E c o 1 )

K ( S f a e )

Glutaraldehyde Cross-link Site of SR Ca2+-ATPase

M T G D G V N D A P A L K K A E I G I A M G S .

VTGDG@IDGPALKKADVGFAMGIA

. . . . . C Q R Q G A I V A V T G D G V N D

. . . . . C Q R L G A I V A V T G D G V N D

. . . . . L Q Q R G Y L V A M T G D G V N D A P ~ L K K A D ~ I A V E G .

. . . . . LRQRGYTCAMTGDGVNDAPALKRADVGIAVHG.

. . . . . Y Q A E G R L V A M T G D G

. . . . . Y L D Q G K K V I M ~ G D G I N D A S L

FIG. 6. Sequence homologies among P-type ATPases in the region around Arg-678. The amino acid sequence of Ca2+-ATPase of sarcoplasmic reticulum from fast twitch rabbit skeletal muscle (labeled Ca(SSR); Ref. 16) is compared with examples of other ATPases: Ca(HPL), Ca’+-ATPase of human plasmalemma (34); Na(Tor), a-subunit of Na+,K+-ATPase of Torpedo californica (35); H(Hgas), H+,K+- ATPase of human gastric mucosal cells (36); H(Neur), H+-ATPase of Neurospora crassa plasmalemma (37); ?(Ldon), ATPase of Leishmania donouani (38); K(Eco0, K+-ATPase of E. coli (39); K(Sfae), K’-ATPase of Staphylococcus fuecalis (40). Arg-678 is shown with a double asterisk. Lys-684 derivatized with pyridoxal-ATP (11) and Lys-712, which is the equivalent residue to Lys-725 of Na+,K+-ATPase derivatized with 5’- (p-fluorosulfony1)benzoyladenosine (30), are each labeled with a single asterisk. Homologous amino acids with the same residue or a conservative replacement in seven out of the eight sequences are boxed together. The sequences were aligned by visual inspection.

W FIG. 7. Two-dimensional representation of the Ca2+-ATP-

ase showing the cross-link site and other residues derivatized by affinity probes in this and other ATPases. The location of the membrane-spanning segments is taken from MacLennan et al. (27). Lys-492 is derivatized with TNP-8N3-AMP and -ATP (9) and pyridoxal-ATP (13) and the equivalent Lys-480 of the lamb kidney Na+,K+-ATPase is derivatized with pyridoxal 5”phosphate and pyr- idoxal-ADP (25). A peptide from the equivalent region of the dog kidney Na+,K+-ATPase is derivatized with 2-azido-ATP (26). Lys- 515 is derivatized with fluorescein 5’-isothiocyanate (41). Residues 623-634 represent the equivalent residues (560-566) of the yeast plasma membrane H+-ATPase derivatized with 2-azido-ATP (28). Lys-684 is derivatized with pyridoxal-ATP (11). Cys-670 and Lys- 712 are the residues equivalent to Cys-662 and Lys-725 of the Na+,K+- ATPase derivatized with 5’-(p-fluorosulfonyl)benzoyladenosine (30).

phorylation site at one apex. Homologies with known ATP binding tertiary structures are tenuous, and the P-type AT- Pases may have a unique structure. There has been an attempt to model the nucleotide binding site on that of adenylate kinase (31), although it is unfortunate that both segments encompassing Lys-492 and Arg-678 are located just outside the region analyzed. Serrano (3) has suggested that the nu- cleotide binding site of the P-type ATPases may be closer to that of phosphofructokinase (3). Placing Lys-492 and Arg- 678 in close proximity restricts the possible permutations but does not appear to favor one model over the other.

Lys-684 is probably in close proximity to Lys-492 and Arg- 678, since mutation of the latter to a histidyl or a glutaminyl residue inhibits the cross-link, although, interestingly, re- placement with an arginyl or valyl has no effect (12). Their proximity to each other is also suggested from the fact that both Lys-492 and Lys-684 are labeled by pyridoxal-ATP in

the absence of Ca2+ (13). Conserved Pro-681 equidistant be- tween Arg-678 and Lys-684 (Fig. 4) may form the apex of a hairpin loop positioning the latter residues alongside each other. Lys-515 is probably also a near neighbor, as derivati- zation with fluorescein isothiocyanate is also inhibitory (4). Asp-627 and Asp-703 of the two conserved triplets Thr-Gly- Asp may be involved in complexing the catalytic M e ion.

A rather unusual feature of the nucleotide binding site is the large number of lysyl and arginyl residues. At least five are implicated a t present (Fig. 7). This concentration of positive charges may play a role in ATP-mediated conforma- tional changes. The catalytic site serves both a catalytic and regulatory function (8, 9). In its function as a catalytic site, there is evidence that nucleotide binding induces a rate- limiting conformational change that facilitates phosphoryla- tion (32). ATP binding could cause such conformational changes in part by neutralizing and shielding electrostatic repulsion between some of these residues. There is evidence that phosphorylation of the cross-linked ATPase by smaller substrates such as acetyl phosphate is accelerated compared with the uncross-linked enzyme (6), perhaps by the cross-link removing the charges on the two linked residues.

Aside from its effect on nucleotide binding, the cross-link completely blocks formation of E2-P (6, 7). In the forward direction of catalysis Ca2+ cannot be released to the vesicle lumen, resulting in a pronounced stabilization of EI-P(2Ca) (Scheme I). Since the cross-link is located at the nucleotide binding site on the opposite side of the membrane to the lumen, it is evident that the block is caused by the cross-link preventing an essential long range conformational change. This suggests that Lys-492 and Arg-678 are at a pivotal position in the active site where domain or segmental move- ments are significant. This conclusion is substantiated by the preferred mutation of these residues to glycyl residues noted above. Several conserved residues, located in various regions of the polypeptide, have been identified by site-directed mu- tagenesis as being essential for proper execution of this con- formational change (12, 29). Serrano and Portillo (33) have suggested on the basis of site-directed mutagenesis studies with the plasma H+-ATPase of Saccharomyces cerevisiae that part of the conformational change involves repositioning of the segment containing the lysyl residue labeled by fluorescein isothiocyanate and a Thr-Gly-Glu-Ser (residues 231-234 in the Ca2+-ATPase) segment closer to the phosphorylation site, since each segment has residues essential for different stages of the catalytic cycle. If Lys-492 is considered an integral part

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Glutaraldehyde Cross-link Site of SR Ca2+-ATPase 22335

of the Lys-515 segment, this could explain why the cross-link is so crippling. A role of the Arg-678 region in the tertiary structural change is suggested from mutations of Lys-684 of the Ca2'-ATPase. Mutation to an arginyl residue caused the enzyme to exhibit similar features to the cross-linked ATPase, in that phosphorylation to EI-P(2Ca) occurred and the sub- sequent conformational change to E*-P was inhibited and phosphorylation by Pi was blocked (12). Mutation to other residues had more serious consequences and blocked phos- phorylation in both directions. The Arg-684 mutant appeared to be able to attain the E*-P conformation although at a much slower rate. It may transpire that both the Lys-492 and Arg- 678 regions move with respect to the phosphorylation site and each other, perhaps to close the active site and communicate with the transport sites.

Acknowledgments-I thank Jeanette Gibson and David G. Woolley for expert technical assistance, Mervyn C . Berman for discussions, and David C . Ross for contributions during the early part of this study.

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D B McIntoshreticulum Ca(2+)-ATPase.

Glutaraldehyde cross-links Lys-492 and Arg-678 at the active site of sarcoplasmic

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