Prorein Science (1998). 7789-793. Cambridge University Press. Printed in the USA Copyright 0 1998 The Protein Society

FOR THE RECORD

Tautomeric state and pK, of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoeno1pyruvate:sugar phosphotransferase system

DANIEL S. GARRETT,' YEONG-JAE SEOK,* ALAN PETERKOFSKY,3 G. MARIUS CLORE,' AND ANGELA M. GRONENBORN' 'Laboratory of Chemical Physics, Building 5 , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520

'Department of Microbiology, College of Natural Sciences, Seoul National University, Seoul 15 1-742, Korea 'Laboratory of Biochemical Genetics, Building 36, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

(RECEIVED November 17, 1997; ACCEPTED December 24, 1997)

Abstract: The phosphorylated form of the N-terminal domain of enzyme I of the phosphoeno1pyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range IH-I5N correlation spectroscopy. The active site His 189 is phosphorylated at the Ne2 position and has a pK, of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the N61-H tautomer, and its Ne2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a x2 angle in the g + conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.

Keywords: histidine phosphorylation; N-terminal domain of en- zyme I; pK,; tautomeric state

Transport of some hexose sugars across the cytoplasmic mem- brane of bacterial cells is coupled to a phosphorylation cascade involving several protein intermediates (see Herzberg & Klevit, 1994 and Postma et al., 1996 for reviews). Escherichia coli

Reprint requests to: G.M. Clore or A.M. Gronenborn, Laboratory of Chemical Physics, Building 5 , National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520; e-mail: clore@vger.niddk.nih.gov or gronenborn@vger.niddk. nih.gov.

Abbreviafions: PTS, phosphoeno1pyruvate:sugar phosphotransferase sys- tem of E. coli; EI, enzyme I of the PTS; EIN, N-terminal domain (residues 1-258 + Arg) of EI; EII, enzymes I1 of the PTS; HPr, histidine-containing phosphocarrier protein of the PTS; PEP, phosphoenolpyruvate; HSQC, heteronuclear single quantum coherence.

Enzyme I, the first protein in the phosphoryltransfer pathway, is autophosphorylated by phosphoenolpyruvate at His 189. Phos- phorylated E1 acts as the phosphoryl donor to His 15 of the histidine-containing phosphocanier protein, HPr. Phosphorylated HPr in turn donates the phosphoryl group to sugar transporters, collectively known as enzymes 11. E1 is a 64-kDa protein con- sisting of N- and C-terminal domains (LiCalsi et al., 1991; Lee et al., 1994). The N-terminal domain of E1 terminates in the linker region from Glu 252 to Leu 264 (Lee et al., 1994) and can be phosphorylated on His 189 in a fully reversible manner by phosphorylated HPr, although it has lost its ability to become phosphorylated by PEP (Chauvin et al., 1996; Seok et al., 1996). In addition, EIN is capable of functioning in phosphotransfer reactions with a variety of acceptor proteins that cannot be phos- phorylated by intact E1 (Seok et al., 1996).

Recently, crystal (Liao et al., 1996) and solution NMR (Garrett et al., 1997a) structures of EIN (1-258 + Arg) have been deter- mined and the interaction surface between EIN and Hpr has been mapped (van Nuland et al., 1995; Garrett et al., 1997b). EIN con- sists of an (Y domain (residues 33-143) and an alp domain (res- idues 1-20 and 148-230) connected by linkers (residues 21-32 and 144-147), and a C-terminal helix (residues 233-250) (Fig. 1). The active site His 189 is located at the interface of the a and alp domains. In the unphosphorylated state, His 189 has a pK, of 6.3 (Garrett et al., 1997a). At neutral pH, His 189 exists as the N61-H tautomer (Garrett et al., 1997a) and its Ne2 atom accepts a hydro- gen bond from the hydroxyl of Thr 168 (Liao et al., 1996; Garrett et al., 1997a). The pKo and tautomeric state of His 189 remain unchanged upon complex formation with HPr (in the absence of phosphorylation) (Garrett et al., 1997b).

Figures 2 and 3 show the one-bond and long-range 'H-I5N correlation spectra, respectively, of I5N-labeled E M at pH 6.9 in an approximately 1: 1 mixture of phosphorylated and unphosphor-

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D.S. Garrett et al.

Fig. 1. Schematic ribbon drawing of EIN showing the location of the active site His 189 at the interface of the P and a@ domains. The location of the shallow depression at the interface of the two domains that provides access to His 189 is indicated by the red arrow. The ribbon is color coded, based on the results of the one-bond 'H-I5N HSQC spectrum of a 1:l mixture of phosphorylated and unphosphorylated EJN (Fig. 2), as follows: yellow, residues whose IH-l5N cross-peaks are clearly split into two cor- responding to phosphorylated and unphosphorylated protein; blue, residues whose 'H-l5N cross-peaks are unaffected by phosphorylation; grey, resi- dues for which we cannot ascertain with certainty whether they are affected or unaffected by phosphorylation owing to spectral overlap. The coordi- nates are taken from Garrett et al. (1997a) and the figure was generated with the program MOLMOL (Koradi et al., 1996).

ylated forms. The phosphorylated and unphosphorylated forms of EIN are in slow exchange on the chemical shift time scale. 'Tko major classes of cross-peaks are observed in the one-bond 'H-"N HSQC spectrum (Fig. 2): those that are split into two correspond- ing to the phosphorylated and unphosphorylated protein, and whose intensities are consequently reduced by a factor of two relative to a sample of unphosphorylated EIN; and those that are unaffected by phosphorylation. The majority of residues whose 'H-''N cross- peaks are perturbed by phosphorylation are located at the interface between the a and a/B domains and within the a@ domain. This primarily reflects the location of the phosphorylated His 189 at the interface of the two domains, protruding upward from the a@ domain toward the a domain (Fig. 1). Interestingly, whereas the backbone NH and "N chemical shifts of a large number of resi- dues in the n/B domain are affected by phosphorylation of His 189, HPr binding results in backbone chemical shift perturba- tions of only a small number of residues in the a / p domain but a large number in the a domain (Garrett et al., 1997b).

The long-range 'H-"N HSQC spectrum (Fig. 3) correlates the imidazole nitrogens with the carbon-attached ring protons of his- tidine. The N d and NSl resonances are readily distinguished be- cause mss-peaks of approximately equal intensity are observed for the N~2-He1, NR-Ha, and NS1-HE1 two-bond correlations (2Jm - 6-10 H z ; Blomberg et al., 1977). whereas either a very weak or absent cross peak is observed for the NSl-HS2 three-bond correlation (3Jm - 2-3 Hz, Blomberg et al., 1977). For a neutral histidine, the protonated ring nitrogen resonates at -168 ppm, whereas the unprotonated nitrogen resonates at -250 ppm (refer- enced relative to the ''N chemical shift of liquid ammonia; Pelton et al., 1993). For a positively charged fully protonated histidine, the two nitrogens resonate around 175 ppm, with the NS1 atom generally resonating about 1 ppm to lower field than the N R atom (Pelton et al., 1993). The pattern and positions of the cross-peaks of His 76, His 97, and His 105 are either unaffected (His 97 and His 105) or minimally perturbed (His 76) upon phosphorylation, indicating that their pK, values (<6, -7.3, and -6.4, respectively) and tautomeric states for the neutral species (Ne2-H tautomer) remain unchanged (Garrett et al., 1997a). In contrast, two distinct sets of cross-peak patterns are observed for His 189. One set, corresponding to the unphosphorylated form, displays a pattern of NSl-Hd, Nd-Hd, and N d - H a cross-peaks, with NSl and Ne2 chemical shifts of 191 and 220 ppm, respectively, characteristic of the neutral N61-H tautomeric state (Garrett et al., 1997a). The second set, corresponding to the phosphorylated form, is charac- terized by a pattern of NS1-Hd, N~2-He1, and NR-HS2 cross- peaks, with NSl and Ne2 chemical shifts of 203 and 207 ppm, respectively.

The expected 15N chemical shifts for a phosphorylated nitro- gen, derived from NMR studies using model compounds such as N-phosphoimidazole and N-methyl-N-phosphoimidazole, are -209 ppm for a neutral phosphoimidazole nitrogen and -202 ppm for the cationic phosphoimidazolium moiety (Pelton et al., 1993). The titration curves for the N d and NSl reso- nances of phosphorylated His 189 are shown in Figure 3B. Both resonances exhibit Henderson-Hasselbach behavior, with a pK, of 7.33 k 0.05. The "N chemical shift of the N d atom varies only minimally with pH, with limiting values of 209.4 ppm at low pH and 200.5 ppm at high pH, typical of a phosphorylated nitrogen. The "N chemical shift of the NS1 atom, on the other hand, ranges from 188 ppm at pH 6.1 to 241.8 ppm at pH 8.3, close to those expected for the protonated and unprotonated states, respectively, of a nonphosphorylated nitrogen in a phosphoimid- azole (Pelton et al., 1993). Thus, we conclude that His 189 is phosphorylated at the Ne2 position, in agreement with previous biochemical results (Weigel et al., 1982).

The pK, of phosphorylated His 189 (-7.3) is one unit higher than that of the unphosphorylated form (-6.3; Garrett et al.. 1997a). Moreover, although the neutral species of unphosphorylated His 189 is in the NS1-H tautomer such that the N d nitrogen becomes protonated at low pH, in phosphorylated His 189, the NE^ atom of His 189 is bonded to the phosphate group and protonation occurs at the NSl atom. Because the Ne2 atom is solvent inacces- sible and accepts a hydrogen bond from the hydroxyl of Thr 168 in unphosphorylated EIN (Garrett et al., 1997a), these results im- ply that both protonation of unphosphorylated His 189 and phos- phorylation of His 189 must be accompanied by a conformational change of the imidazole ring. In unphosphorylated EIN, the x1 and x2 angles of His 189 are in the trans and g+ conformations, respectively (Liao et al., 1996; Garrett et al., 1997a). Because the

Active site histidine of phospholylated EIN 79 1

Dl01 0

n o 9 at

G206 0

N224 0

I1W 0

NA30

0 1 O/

15 0 L220

I147 0 * A !

v25 - 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5

100

110

120

.130

'H (PPW

Fig. 2. One-bond 'H-I5N HSQC spectrum of an approximately 1: 1 mixture of phosphorylated and unphosphorylated I5N-labeled EIN at 30°C and pH 6.9. Some of the cross-peaks whose intensity are reduced by half upon phosphorylation owing to splitting into two cross-peaks corresponding to the phosphorylated and unphosphorylated states are labeled in bold. In those cases where the cross-peaks in the phosphorylated state are assigned, an arrow from the cross-peak of the unphosphorylated state to that of the phosphorylated state is shown. Some of the cross-peaks whose intensities are either unaffected or minimally affected upon phosphorylation are shown in italics. The two boxed regions indicate the location of the cross-peaks of Glu 68 and His 189 in the unphosphorylated state. Assignments of the unphosphorylated state are taken from Garrett et al. (1997a).

phosphorylated Ne2 atom must become accessible to HPr and because the active site His 15 of HPr approaches His 189 along the shallow depression at the interface of the (Y and (Y@ domains (Garrett et al., 1997b; Fig. l), the conformational change must involve a change in the x2 angle of His 189 from the gf to the g- conformation (Liao et al., 1996; Garrett et al., 1997a, 1997b). Interestingly, His 189 phosphorylation is accompanied by a sub- stantial decrease in thermal stability of EIN (N.J. Nosworthy, A. Peterkofsky, S. Konig, Y.-J. Seok, R.H. Szczepanowski, &A. Gins- burg, private comm.).

Figure 4 presents a schematic view of some of the structural determinants in the phosphorylation reaction of EL At physiolog- ical pH (-7), unphosphorylated His 189 is predominantly in the neutral NSl-H tautomeric state with the unprotonated Ne2 atom accepting a hydrogen bond from Thr 168. For phosphorylation to occur, the imidazole ring has to flip about x 2 and a similar ring flip has to occur upon protonation. The latter has to be fast (> 1.5 X los s-' - 2rA, where A is the difference in "N chemical shift of the Ne2 atom in the protonated and unprotonated states of unphos- phorylated EIN) because the neutral and protonated forms of His 189 are in fast exchange on the NMR time scale, and it may well be the case that phosphorylation occurs on the protonated

imidazole ring on which the Ne2 edge of His 189 is exposed. Indeed, at pH -7, the phosphohistidine is predominately proton- ated. Stabilization of the phosphorylated form of E1 could be ac- complished by ion-pair interaction of the phosphoryl group with the NH: group of Lys 69, which is -5 8, away from the imidazole ring of His 189. This is supported by the observation of large backbone chemical shift observed in the one-bond 'H-I'N HSQC spectrum upon phosphorylation involving residues Phe 65 to Glu 70.

Materials and methods: Phosphorylation of EIN: Enzyme I, "N EIN, and HPr were prepared as described by Reddy et al. (1991) and Garrett et al. (1997a, 1997b). respectively. Phosphorylation of EIN was performed in a reaction mixture (350 pL) contain- ing 1.5 mM uniformly (>98%) "N-labeled EIN, 1 p M HPr, 15 or 23 nM EI, 100 mM sodium phosphate buffer, pH 7.2, 2 mM MgC12, 2 mM DTT, and 50 mh4 PEP. A pilot experiment using 32P-PEP showed that, under these conditions, EIN was main- tained in the phosphorylated form between 20 and 58 h after the addition of PEP at 37°C. With 15 nM E1 in the reaction mix- ture, EIN was -50% phosphorylated as judged by NMR; in

792 D.S. Garrett et al.

A 1604 NEZHEi creasing the concentration of E1 to 23 nM resulted in >90%

- 180-

-

200-

-

220-

-

240-

His97

N6

NSI-HE

N

8.5 8.0 715 7.0 6.5 6.0 'H (ppm)

190- NE2

200 - a .

210- - L

220

240

6.0 6.5 7.0 7.5 8.0 8.5 PH

Fig. 3. A: Long-range 'H-I'N HSQC spectrum of an approximately 1: 1 mix- ture of phosphorylated and unphosphorylated "N-labeled EIN at 40 "C and pH 6.9. Two distinct sets of cross-peaks are observed for His 189, corre- sponding to the phosphorylated and unphosphorylated states of His 189; the resonances of His 76 are minimally perturbed upon phosphorylation with the Ne2 resonance slightly downfield, the He1 resonance slightly upfield, and the H62 resonance slightly downfield, relative to the unphosphorylated EIN; the resonances of His 97 and His 105 are unperturbed by phosphor- ylation. B: Titration of the Ne2 and N61 resonances of phosphorylated His 189 obtained by recording a series of long-range 'H-I5N correlation spec- tra at 30 "C on a sample containing 290% phosphorylated EIN. The con- tinuous lines represent the best-fit curves for a single pK,, with a pKa value of 7.33 + 0.05, and N61 and Ne2 "N chemical shifts of 184.9 and 209.4 ppm, respectively, for the fully protonated state, and 247.8 and 200.5 ppm, respectively, for the fully unprotonated neutral state. "N chem- ical shifts are referenced relative to liquid ammonia (Pelton et al., 1993).

phosphorylation of EIN.

NMR spectroscopy: NMR experiments were performed at 30°C or 40°C on a Bruker AMX600 spectrometer equipped with a z-shielded gradient triple resonance probe. Two-dimensional 'H-I5N HSQC spectra were recorded using a water flip-back as described by Grzesiek and Bax (1993). Two-dimensional long- range 'H-I5N HSQC spectra to correlate the NS1 and Ne2 ring nitrogens with the CElH and CS2H ring protons of histidine were recorded with a 22-ms dephasing delay, during which the 'H and I5N signals become antiphase (Pelton et al., 1993). Spec- tra were processed with the NmrPipe package (Delaglio et al., 1995) and analyzed using the programs PIPP, CAPP, and STAPP (Garrett et al., 1991).

Acknowledgments: We thank Frank Delaglio for software and Rolf Tschudin for technical support.

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El-His 189 I

El-HIS 189

Fig, 4. Schematic of the phosphorylation reaction of EIN. In the unphosphorylated state, the x2 side-chain torsion angle of His 189 is in the g+ conformation, and the Ne2 atom of His 189 is solvent inaccessible and accepts a hydrogen bond from the hydroxyl of Thr 168. In the phosphorylated state, the x2 angle of His 189 is in the g- conformation, the Ne2 atom is directly bonded to the phosphate, and the negative charge on the phosphate is stabilized by an ion-pair interaction with the side chain of Lys 69. The state of His 189 depicted in the figure represents the predominant form at pH 7.

Active site histidine of phosphorylated EIN 793

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