- Email: [email protected]
Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation
BBRC Biochemical and Biophysical Research Communications 350 (2006) 1044–1049 www.elsevier.com/locate/ybbrc
Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation q Jun Hyuck Lee a, Young Jun Im a, Jungdon Bae b, Dooil Kim b, Mun-Kyoung Kim a, Gil Bu Kang a, Dae-Sil Lee b, Soo Hyun Eom a,* a
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea b Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea Received 18 September 2006 Available online 6 October 2006
Abstract Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs ˚ crystal structure of unliganded PGK from Thermus that brings the N- and C-domains of PGK closer together. Here we present the 1.8 A caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Crystal structure; Domain movement; Phosphoglycerate kinase; Thermus caldophilus
The glycolytic enzyme phosphoglycerate kinase (PGK) catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP, yielding 3-phosphoglycerate (3-PG) and ATP. PGK is a monomeric enzyme comprised of globular N- and C-domains connected by a narrow hinge region; the active site is located at the interface between the two domains. Substrate 1,3-BPG binds to the N-domain of PGK, while ADP binds to the C-domain. This substrate binding leads to a conformational change that closes the active-site cleft; i.e., the catalytic mechanism is associated with a bending motion at the hinge, which brings the two substrates into close proximity q Abbreviations: 3-PG, 3-phosphoglycerate; 1,3-BPG, 1,3-bisphosphoglycerate; AMPPNP, b,c-imido-adenosin-5 0 -triphosphate; AMPPCP, b,cmethylene-adenosine-5 0 -triphosphate; PGK, phosphoglycerate kinase; RMSD, root mean square deviation. * Corresponding author. Fax: +82 62 970 2548. E-mail address: [email protected] (S.H. Eom).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.151
[1,2]. Such domain closure can occur only when the enzyme is in a ternary complex with its two substrates; it does not occur when the enzyme is a binary complex with either one of its substrates [3]. X-ray crystallographic studies of PGKs have contributed significantly to our understanding of the hinge closure mechanism. In several crystal structures, the domains of PGK occupy somewhat different positions, and we have divided the observed conformations into two groups: open and closed. In some cases, however (e.g., pig muscle PGK), large-scale domain movements are apparently prevented by the crystal lattice forces [4]. For instance, the structures of PGK from pig muscle (PDB code 1HDI, ADP, and 3-PG complex structure [5]; PDB code 1VJC, MgATP complex structure [6]; PDB code 1VJD, ATP complex structure [6]; PDB code 1KF0, AMPPCP, and 3-PG complex structure [7]), yeast (PDB code 3PGK, ATP, and 3-PG complex structure [8]), and Bacillus stearothermophilus (PDB code 1PHP,
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049 Table 1 Data collection and refinement statistics Data collection X-ray source Space group ˚) Unit-cell parameters (A ˚) Wavelength (A ˚) Resolution (A Observed reflections Unique reflections Rmerge (%)a Average I/r Data coverage total/final shell (%) Redundancy
PF-18B P212121 a = 65.1, b = 71.3, c = 80.2 1.000 29.4–1.8 (1.92–1.8) 301,816 34,829 5.9 (32.1) 8.5 (2.3) 99.9/99.9 3.9
Refinement ˚) Resolution (A Number of residues Number of water molecules Rcryst (%)b Rfree (%)c ˚) RMSD bond length (A RMSD bond angle (°) ˚ 2) Average B value (A
20–1.8 390 178 20.9 27.3 0.006 1.37 32.9
1045
3-PG complex [3]; PDB code 16PK, bisubstrate analogue: partially closed structure [11]) have closed confirmations. No open (apo) structures of PGKs from Thermotoga maritima (Tma) and Trypanosoma brucei (Tbr) have been reported so far. To better understand the motion of the PGK domains, ˚ resoluwe have solved the structure of TcaPGK at 1.8 A tion in the absence of substrate. Because TcaPGK (open conformation) and TmaPGK (closed conformation) show 58.3% amino acid sequence identity, comparison of these two structures enables detailed explanation of the hinge bending motion responsible for the open-to-closed transition. Materials and methods
Values in parentheses refer to the highest resolution shell. P P a Rmerge ¼ jhIi Ij= hIi. P P b Rcryst ¼ jjF o j jF c jj= jF o j. c Rfree calculated with 10% of all reflections excluded from refinement stages using high resolution data.
ADP complex structure [9]) have open conformations, whereas the structures of PGK from Thermotoga maritima (PDB code 1VPE, AMPPNP, and 3-PG complex structure [10]) and Trypanosoma brucei (PDB code 13PK, ADP, and
Protein purification, crystallization, and data collection. Overexpression and purification of TcaPGK, its crystallization, and X-ray data collection have been described previously [12]. Briefly, the gene encoding TcaPGK was subcloned between the EcoRI and PstI sites of the expression vector pKK223-3 (Pharmacia Biotech), which was then used to transform Escherichia coli strain JM109. The recombinant TcaPGK protein was purified by heat treatment, ion exchange chromatography (DEAE– Sepharose column), and size exclusion chromatography (Superdex 200 column). Crystals were grown by equilibrating a mixture containing 1 ll of protein solution [13 mg ml1 protein in 20 mM Hepes–NaOH (pH 7.5), 150 mM KCl] and 1 ll of reservoir solution [24% (w/v) PEG 4000, 0.1 M sodium citrate, pH 6.0, and 0.2 M ammonium acetate] against 0.5 ml of reservoir solution. A set of native data was collected to a resolution of ˚ at beam line BL-18B at the Photon Factory, Japan, using an X-ray 1.8 A ˚. beam with a wavelength of 1.0000 A Structure determination and refinement. The structure of TcaPGK was determined by molecular replacement using the program MOL-
Table 2 Known PGK crystal structures and analysis of domain movement PDB code
TmaPGK
BstPGK
TbrPGK
PigPGK
YeastPGK
1VPE
1PHP
13PK
16PK
1HDI
1VJC
1VJD
1KF0
3PGK
Reference
[10]
[9]
[3]
[11]
[5]
[6]
[6]
[7]
[8]
Domain conformation
Closed
Partially closed
Closed
Partially closed
Open
Open
Open
Open
Open
Sequence identity/ homology with TcaPGK (%)
58/74
50/68
42/61
42/61
38/56
38/56
38/56
38/56
38/56
Complexed ligand
Mg-AMPPNP, 3-PG
Mg-ADP
Mg-ADP, 3-PG
Bisubstrate analoguea
Mg-ADP, 3-PG
Mg-ATP
ATP
MgAMPPCP, 3-PG
Mg-ATP, 3-PG
(1) 15.8 (2) 0.4
(1) 15.1 (2) 0.1
(1) 14.2 (2) 1.0
NDc
ND
ND
ND
ND
ND
175–176, 202–203, 206–207, 365–366
167–168, 369–370, 377–382
177–178, 193–201, 208–209, 359–360, 372–376
Domain movement (fixed N-domain of TcaPGK)b (1) Rotation angle (°) ˚) (2) Translation (A Hinge region residuesd
a
Adenyl 1,1,5,5-tetrafluoropentane-1,5-bisphosphonate. When the N-domain of TcaPGK was superimposed onto that of other PGK structures, the movement of the C-domain was analyzed using the program DynDom [16]. c ND, domain movement was not detected. d Residue numbering corresponds to the amino acid sequence of TcaPGK. b
1046
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049
REP [13]. For the cross-rotation search, the structure of TmaPGK (PDB code 1VPE) [10] was used as the search model, and the highest peak of the rotation function was used for the translation function. This model gave a strong single peak in the translation function. The structure of initial molecular replacement model was refined as two rigid bodies corresponding to the N- and C-domains, using the program CNS [14]. Subsequent simulated annealing, overall anisotropic Bfactor, and individual restrained B-factor refinements were also carried out using CNS. After these refinement steps, an interpretable electron density map was calculated. Many cycles of manual rebuilding, using the program O [15] and refinement using CNS, yielded a final crys-
tallographic R value of 20.9% (Rfree = 27.3%). Atomic coordinates have been deposited at the Protein Data Bank under accession code 2IE8. Analysis of the domain motion. Two PGK structures were used in this ˚ resolution TmaPGK structure (PDB code 1VPE; closed analysis: the 2.0 A conformation) and the substrate-free TcaPGK structure (reported here; open conformation). The program DynDom [16] was used to analyze the motion of the domains, using a sliding five-residue window. The minimum value for the ratio of the interdomain displacement was set to 1, and the minimum domain size was 10 residues. Hinge residues were identified at the transition point between the two dynamic domains.
Fig. 1. Crystal structure of TcaPGK. (A) Ribbon diagram showing the overall structure of TcaPGK protein. The a-helices are labeled a1–a17 and b-strands are labeled b1–b15. (B) Multiple sequence alignment of PGKs from Thermus caldophilus (TcaPGK), Thermotoga maritima (TmaPGK), Bacillus stearothermophilus (BstPGK), Trypanosoma brucei (TbrPGK), pig muscle (PigPGK), and yeast (YeastPGK) carried out using ClustalX [18]. Highly conserved residues are shaded in black and gray. Interdomain salt bridge forming residues (R60, R148, D197, and E375) are shown in blue. * indicates the intervals of 10 amino acids.
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049
Results and discussion Overall structure of TcaPGK The crystal structure of TcaPGK was solved by molecu˚ resolution. The lar replacement and then refined to 1.8 A Ramachandran plot calculated with the program PROCHECK [17] showed no residues with angular values in disallowed areas: 97% of residues were in the most favored regions, while 3% were in allowed regions. The crystals belong to the P212121 space group with one molecule per asymmetric unit. The final model of TcaPGK included one protein molecule (amino acid residues 1–390) and 178 water molecules. Data collection and refinement statistics are summarized in Table 1. The overall topology of the TcaPGK is very similar to that described for the enzymes from pig muscle, yeast, Bacillus stearothermophilus, Thermotoga maritima, and Trypanosoma brucei (Table 2). The structure of the TcaPGK is comprised of 15 b-strands (b1–b15) and 17 ahelices (a1–a17), and can be divided into two domains: an N-domain (residues 1–169 plus residues 382–390) and a C-domain (residues 167–381) (Fig. 1A). The 3-PG-binding site is situated in the N-domain, while the nucleotide binding site is in the C-domain. The C-terminus crosses back to the N-domain. Comparison with other PGK structures Structural superposition of the TcaPGK apo structure (open conformation) and TmaPGK structure (closed conformation) in complex with AMPPNP/3-PG (PDB code ˚ for 296 Ca atoms. Compar1VPE) gave a RMSD of 1.7 A isons of the respective N- and C-domains within these two ˚ for 170 Ca atoms structures yielded RMSD values of 0.9 A ˚ in the N-domain (residues 1–169 plus 382–390) and 1.2 A for 194 Ca atoms in the C-domain (residues 167–381) (residue numbers in TcaPGK) (Fig. 1B). When the N-domain
1047
of TcaPGK was superimposed onto that of TmaPGK (PDB code 1VPE), a 15.8° rotation of the C-domain (determined using the program DynDom [16]) was observed in the hinge region, which was comprised of residues 175–176, 202–203, 206–207, and 365–366 (residue numbers in TcaPGK) (Fig. 2). By comparing the open and closed structures of apo TcaPGK and TmaPGK, respectively, we are able to determine the features essential for the conformational change of PGK. In the open conformation, an interdomain salt bridge between residues Arg148 in the N-domain and Glu375 in the C-domain forms a strap that ties the two domains together. In the closed conformation, Arg148 (Arg151 in TmaPGK) interacts with the phosphate group of 3-PG, not with Glu375 (Glu381 in TmaPGK); i.e., a change in the orientation of Arg148 enables it to switch from forming an interdomain salt bridge with Glu375 (open conformation) to forming a new intradomain salt bridge with the phosphate group of 3-PG bound (closed conformation). In addition, Arg60 in the N-domain (Arg62 in TmaPGK) forms a new interdomain salt bridge with Asp197 in the C-domain (Asp200 in TmaPGK), which stabilizes the closed conformation. By contrast, Arg60 participates in no direct interactions within the open structure of TcaPGK. Beyond the Ca the Arg60 residue shows little density within the structure of TcaPGK, which implies that Arg60 is intrinsically flexible in the absence of substrate (overall B factor val˚ 2, B factor value of Arg60 ue of TcaPGK structure: 32.9 A 2 ˚ ). Likewise, Asp197 also appears to particiresidue: 43.6 A pate in no direct interactions within the open structure of TcaPGK. Notably, the four residues involved in the salt bridge switch are strictly conserved in all known prokaryotic and eukaryotic PGK sequences (e.g., Fig. 1B). Moreover, it appears that it is reorientation of the interdomain salt bridges after substrate binding that brings the two domains into closer proximity (Fig. 3A and B). Analyses of the nine reported PGK structures revealed that Arg60-Asp197-like salt bridges are observed only
Fig. 2. Comparison of the open and closed conformations of PGK. Shown is a stereo view of the superposition of the structures of TcaPGK (open conformation: slate blue) and TmaPGK (closed conformation: orange) complexed with AMPPNP and 3-PG. Rotation (about 15.8°) of the C-domain may induce the closed conformation. TcaPGK residue numbers are shown in blue, while TmaPGK residue numbers are shown in orange. The figures were made using Pymol [19].
1048
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049
Fig. 3. Domain movement, including interdomain salt bridge switching. (A) Stereo view of the interdomain interactions within the structures of TcaPGK (open conformation: slate blue) and TmaPGK (closed conformation: orange). TcaPGK residue numbers are shown in blue, while TmaPGK residue numbers are shown in orange. (B) Electron density map (2Fo Fc map contoured at 1d) around the Arg148-Glu375 salt bridge within TcaPGK. (C) Salt bridge switching during PGK domain closure is depicted in a cartoon model.
within closed PGK structures (ternary complex: PDB code 1VPE and 13PK) [3,10], and that Arg148-Glu375-like salt bridges are observed within partially closed structures (PDB code 1PHP and 16PK) complexed with ADP or bisubstrate analogue [9,11]. This suggests that nucleotide binding alone is not sufficient to break the Arg148Glu375 salt bridge. On the other hand, 3-PG-complexed structures have no Arg148-Glu375-like salt bridges, although they still resemble the open conformation (PDB code 1HDI, 1KF0, and 3PGK) [5,7,8]. Most likely the binding of 3-PG disrupts the Arg148-Glu375 salt bridge by enabling formation of a new salt bridge between Arg148 and the phosphate group of 3-PG, which is observed in all known 3-PG-complexed structures. In summary, we have resolved the structure of TcaPGK in the absence of substrates (open conformation). Comparison of this open structure with a closed one (TmaPGK) revealed substrate-induced conformational changes, including interdomain salt bridge switching. The formation of an interdomain salt bridge between Arg148 and Glu375 stabilizes the open conformation. The binding of 3-PG disrupts that salt bridge and enables the formation of a new
interdomain salt bridge between Arg60 and Asp197, which stabilizes the closed conformation (Fig. 3C). Acknowledgments We thank Professor N. Sakabe and Drs. N. Igarashi and N. Matsugaki for their kind support during data collection at BL18B of the Photon Factory (Tsukuba, Japan). This research was supported by a grant from the KRIBB Research Initiative Program. References [1] R.D. Banks, C.C.F. Blake, P.R. Evans, R. Haser, D.W. Rice, G.W. Hardy, M. Merrett, A.W. Phillips, Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme, Nature 279 (1979) 773–777. [2] C.C. Blake, D.W. Rice, Phosphoglycerate kinase, Phil. Trans. Soc. Ser. A 293 (1981) 93–104. [3] B.E. Bernstein, P.A. Michels, W.G. Hol, Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation, Nature 385 (1997) 275–278.
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049 [4] Z. Kovari, M. Vas, Protein conformer selection by sequencedependent packing contacts in crystals of 3-phosphoglycerate kinase, Proteins 55 (2004) 198–209. ˚ resolution [5] A.N. Szilagyi, M. Ghosh, E. Garman, M. Vas, A 1.8 A structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and 3-phosphoglycerate in open conformation: new insight into the role of the nucleotide in domain closure, J. Mol. Biol. 306 (2001) 499–511. [6] B. Flachner, Z. Kovari, A. Varga, Z. Gugolya, F. Vonderviszt, G. Naray-Szabo, M. Vas, Role of phosphate chain mobility of MgATP in completing the 3-phosphoglycerate kinase catalytic site: binding, kinetic, and crystallographic studies with ATP and MgATP, Biochemistry 43 (2004) 3436–3449. [7] Z. Kovari, B. Flachner, G. Naray-Szabo, M. Vas, Crystallographic and thiol-reactivity studies on the complex of pig muscle phosphoglycerate kinase with ATP analogues: correlation between nucleotide binding mode and helix flexibility, Biochemistry 41 (2002) 8796–8806. [8] H.C. Watson, N.P. Walker, P.J. Shaw, T.N. Bryant, P.L. Wendell, L.A. Fothergill, R.E. Perkins, S.C. Conroy, M.J. Dobson, M.F. Tuite, A.J. Kingsman, S.M. Kingsman, Sequence and structure of yeast phosphoglycerate kinase, EMBO J. 1 (1982) 1635–1640. [9] G.J. Davies, S.J. Gamblin, J.A. Littlechild, Z. Dauter, K.S. Wilson, H.C. Watson, Structure of the ADP complex of the 3-phosphoglyc˚ , Acta Cryserate kinase from Bacillus stearothermophilus at 1.65 A tallogr. D Biol. Crystallogr. 50 (1994) 202–209. [10] G. Auerbach, R. Huber, M. Grattinger, K. Zaiss, H. Schurig, R. Jaenicke, U. Jacob, Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability, Structure 5 (1997) 1475–1483. [11] B.E. Bernstein, D.M. Williams, J.C. Bressi, P. Kuhn, M.H. Gelb, G.M. Blackburn, W.G. Hol, A bisubstrate analog induces unexpected
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
1049
conformational changes in phosphoglycerate kinase from Trypanosoma brucei, J. Mol. Biol. 279 (1998) 1137–1148. J.H. Lee, Y.J. Im, S-H. Rho, S.H. Park, G.B. Kang, S.J. Cho, T.-Y. Kim, H-J. Shin, D.-S. Lee, S.H. Eom, Crystallization and preliminary X-ray crystallographic studies of phosphoglycerate kinase from Thermus caldophilus, Mol. Cells 15 (2003) 370–372. A. Vagin, A. Teplyakov, MOLREP: an automated program for molecular replacement, J. Appl. Cryst. 30 (1997) 1022–1025. A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. GrosseKunstleve, J-S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography and NMR system: a new software suite for macromolecular structure determination, Acta Crystallogr. Sect. D Biol. Crystallogr. 54 (1998) 905–921. T.A. Jones, J.Y. Zhou, S.W. Cowan, M. Kjeldgaard, Improved methods for building protein models in electron density maps and the location of errors in these models, Acta Crystallogr. A 47 (1991) 110– 119. S. Hayward, H.J.C. Berendsen, Systematic analysis of domain motions in proteins from conformational change; new results on citrate synthase and T4 lysozyme, Proteins 30 (1998) 144– 154. R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK—a program to check the stereochemical quality of protein structures, J. Appl. Cryst. 26 (1993) 283–291. J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res. 24 (1997) 4876–4882. W.L. DeLano, The PyMOL User’s Manual, DeLano Scientific, San Carlos, CA, 2002.
Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation q Jun Hyuck Lee a, Young Jun Im a, Jungdon Bae b, Dooil Kim b, Mun-Kyoung Kim a, Gil Bu Kang a, Dae-Sil Lee b, Soo Hyun Eom a,* a
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea b Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea Received 18 September 2006 Available online 6 October 2006
Abstract Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs ˚ crystal structure of unliganded PGK from Thermus that brings the N- and C-domains of PGK closer together. Here we present the 1.8 A caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Crystal structure; Domain movement; Phosphoglycerate kinase; Thermus caldophilus
The glycolytic enzyme phosphoglycerate kinase (PGK) catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP, yielding 3-phosphoglycerate (3-PG) and ATP. PGK is a monomeric enzyme comprised of globular N- and C-domains connected by a narrow hinge region; the active site is located at the interface between the two domains. Substrate 1,3-BPG binds to the N-domain of PGK, while ADP binds to the C-domain. This substrate binding leads to a conformational change that closes the active-site cleft; i.e., the catalytic mechanism is associated with a bending motion at the hinge, which brings the two substrates into close proximity q Abbreviations: 3-PG, 3-phosphoglycerate; 1,3-BPG, 1,3-bisphosphoglycerate; AMPPNP, b,c-imido-adenosin-5 0 -triphosphate; AMPPCP, b,cmethylene-adenosine-5 0 -triphosphate; PGK, phosphoglycerate kinase; RMSD, root mean square deviation. * Corresponding author. Fax: +82 62 970 2548. E-mail address: [email protected] (S.H. Eom).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.151
[1,2]. Such domain closure can occur only when the enzyme is in a ternary complex with its two substrates; it does not occur when the enzyme is a binary complex with either one of its substrates [3]. X-ray crystallographic studies of PGKs have contributed significantly to our understanding of the hinge closure mechanism. In several crystal structures, the domains of PGK occupy somewhat different positions, and we have divided the observed conformations into two groups: open and closed. In some cases, however (e.g., pig muscle PGK), large-scale domain movements are apparently prevented by the crystal lattice forces [4]. For instance, the structures of PGK from pig muscle (PDB code 1HDI, ADP, and 3-PG complex structure [5]; PDB code 1VJC, MgATP complex structure [6]; PDB code 1VJD, ATP complex structure [6]; PDB code 1KF0, AMPPCP, and 3-PG complex structure [7]), yeast (PDB code 3PGK, ATP, and 3-PG complex structure [8]), and Bacillus stearothermophilus (PDB code 1PHP,
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049 Table 1 Data collection and refinement statistics Data collection X-ray source Space group ˚) Unit-cell parameters (A ˚) Wavelength (A ˚) Resolution (A Observed reflections Unique reflections Rmerge (%)a Average I/r Data coverage total/final shell (%) Redundancy
PF-18B P212121 a = 65.1, b = 71.3, c = 80.2 1.000 29.4–1.8 (1.92–1.8) 301,816 34,829 5.9 (32.1) 8.5 (2.3) 99.9/99.9 3.9
Refinement ˚) Resolution (A Number of residues Number of water molecules Rcryst (%)b Rfree (%)c ˚) RMSD bond length (A RMSD bond angle (°) ˚ 2) Average B value (A
20–1.8 390 178 20.9 27.3 0.006 1.37 32.9
1045
3-PG complex [3]; PDB code 16PK, bisubstrate analogue: partially closed structure [11]) have closed confirmations. No open (apo) structures of PGKs from Thermotoga maritima (Tma) and Trypanosoma brucei (Tbr) have been reported so far. To better understand the motion of the PGK domains, ˚ resoluwe have solved the structure of TcaPGK at 1.8 A tion in the absence of substrate. Because TcaPGK (open conformation) and TmaPGK (closed conformation) show 58.3% amino acid sequence identity, comparison of these two structures enables detailed explanation of the hinge bending motion responsible for the open-to-closed transition. Materials and methods
Values in parentheses refer to the highest resolution shell. P P a Rmerge ¼ jhIi Ij= hIi. P P b Rcryst ¼ jjF o j jF c jj= jF o j. c Rfree calculated with 10% of all reflections excluded from refinement stages using high resolution data.
ADP complex structure [9]) have open conformations, whereas the structures of PGK from Thermotoga maritima (PDB code 1VPE, AMPPNP, and 3-PG complex structure [10]) and Trypanosoma brucei (PDB code 13PK, ADP, and
Protein purification, crystallization, and data collection. Overexpression and purification of TcaPGK, its crystallization, and X-ray data collection have been described previously [12]. Briefly, the gene encoding TcaPGK was subcloned between the EcoRI and PstI sites of the expression vector pKK223-3 (Pharmacia Biotech), which was then used to transform Escherichia coli strain JM109. The recombinant TcaPGK protein was purified by heat treatment, ion exchange chromatography (DEAE– Sepharose column), and size exclusion chromatography (Superdex 200 column). Crystals were grown by equilibrating a mixture containing 1 ll of protein solution [13 mg ml1 protein in 20 mM Hepes–NaOH (pH 7.5), 150 mM KCl] and 1 ll of reservoir solution [24% (w/v) PEG 4000, 0.1 M sodium citrate, pH 6.0, and 0.2 M ammonium acetate] against 0.5 ml of reservoir solution. A set of native data was collected to a resolution of ˚ at beam line BL-18B at the Photon Factory, Japan, using an X-ray 1.8 A ˚. beam with a wavelength of 1.0000 A Structure determination and refinement. The structure of TcaPGK was determined by molecular replacement using the program MOL-
Table 2 Known PGK crystal structures and analysis of domain movement PDB code
TmaPGK
BstPGK
TbrPGK
PigPGK
YeastPGK
1VPE
1PHP
13PK
16PK
1HDI
1VJC
1VJD
1KF0
3PGK
Reference
[10]
[9]
[3]
[11]
[5]
[6]
[6]
[7]
[8]
Domain conformation
Closed
Partially closed
Closed
Partially closed
Open
Open
Open
Open
Open
Sequence identity/ homology with TcaPGK (%)
58/74
50/68
42/61
42/61
38/56
38/56
38/56
38/56
38/56
Complexed ligand
Mg-AMPPNP, 3-PG
Mg-ADP
Mg-ADP, 3-PG
Bisubstrate analoguea
Mg-ADP, 3-PG
Mg-ATP
ATP
MgAMPPCP, 3-PG
Mg-ATP, 3-PG
(1) 15.8 (2) 0.4
(1) 15.1 (2) 0.1
(1) 14.2 (2) 1.0
NDc
ND
ND
ND
ND
ND
175–176, 202–203, 206–207, 365–366
167–168, 369–370, 377–382
177–178, 193–201, 208–209, 359–360, 372–376
Domain movement (fixed N-domain of TcaPGK)b (1) Rotation angle (°) ˚) (2) Translation (A Hinge region residuesd
a
Adenyl 1,1,5,5-tetrafluoropentane-1,5-bisphosphonate. When the N-domain of TcaPGK was superimposed onto that of other PGK structures, the movement of the C-domain was analyzed using the program DynDom [16]. c ND, domain movement was not detected. d Residue numbering corresponds to the amino acid sequence of TcaPGK. b
1046
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049
REP [13]. For the cross-rotation search, the structure of TmaPGK (PDB code 1VPE) [10] was used as the search model, and the highest peak of the rotation function was used for the translation function. This model gave a strong single peak in the translation function. The structure of initial molecular replacement model was refined as two rigid bodies corresponding to the N- and C-domains, using the program CNS [14]. Subsequent simulated annealing, overall anisotropic Bfactor, and individual restrained B-factor refinements were also carried out using CNS. After these refinement steps, an interpretable electron density map was calculated. Many cycles of manual rebuilding, using the program O [15] and refinement using CNS, yielded a final crys-
tallographic R value of 20.9% (Rfree = 27.3%). Atomic coordinates have been deposited at the Protein Data Bank under accession code 2IE8. Analysis of the domain motion. Two PGK structures were used in this ˚ resolution TmaPGK structure (PDB code 1VPE; closed analysis: the 2.0 A conformation) and the substrate-free TcaPGK structure (reported here; open conformation). The program DynDom [16] was used to analyze the motion of the domains, using a sliding five-residue window. The minimum value for the ratio of the interdomain displacement was set to 1, and the minimum domain size was 10 residues. Hinge residues were identified at the transition point between the two dynamic domains.
Fig. 1. Crystal structure of TcaPGK. (A) Ribbon diagram showing the overall structure of TcaPGK protein. The a-helices are labeled a1–a17 and b-strands are labeled b1–b15. (B) Multiple sequence alignment of PGKs from Thermus caldophilus (TcaPGK), Thermotoga maritima (TmaPGK), Bacillus stearothermophilus (BstPGK), Trypanosoma brucei (TbrPGK), pig muscle (PigPGK), and yeast (YeastPGK) carried out using ClustalX [18]. Highly conserved residues are shaded in black and gray. Interdomain salt bridge forming residues (R60, R148, D197, and E375) are shown in blue. * indicates the intervals of 10 amino acids.
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049
Results and discussion Overall structure of TcaPGK The crystal structure of TcaPGK was solved by molecu˚ resolution. The lar replacement and then refined to 1.8 A Ramachandran plot calculated with the program PROCHECK [17] showed no residues with angular values in disallowed areas: 97% of residues were in the most favored regions, while 3% were in allowed regions. The crystals belong to the P212121 space group with one molecule per asymmetric unit. The final model of TcaPGK included one protein molecule (amino acid residues 1–390) and 178 water molecules. Data collection and refinement statistics are summarized in Table 1. The overall topology of the TcaPGK is very similar to that described for the enzymes from pig muscle, yeast, Bacillus stearothermophilus, Thermotoga maritima, and Trypanosoma brucei (Table 2). The structure of the TcaPGK is comprised of 15 b-strands (b1–b15) and 17 ahelices (a1–a17), and can be divided into two domains: an N-domain (residues 1–169 plus residues 382–390) and a C-domain (residues 167–381) (Fig. 1A). The 3-PG-binding site is situated in the N-domain, while the nucleotide binding site is in the C-domain. The C-terminus crosses back to the N-domain. Comparison with other PGK structures Structural superposition of the TcaPGK apo structure (open conformation) and TmaPGK structure (closed conformation) in complex with AMPPNP/3-PG (PDB code ˚ for 296 Ca atoms. Compar1VPE) gave a RMSD of 1.7 A isons of the respective N- and C-domains within these two ˚ for 170 Ca atoms structures yielded RMSD values of 0.9 A ˚ in the N-domain (residues 1–169 plus 382–390) and 1.2 A for 194 Ca atoms in the C-domain (residues 167–381) (residue numbers in TcaPGK) (Fig. 1B). When the N-domain
1047
of TcaPGK was superimposed onto that of TmaPGK (PDB code 1VPE), a 15.8° rotation of the C-domain (determined using the program DynDom [16]) was observed in the hinge region, which was comprised of residues 175–176, 202–203, 206–207, and 365–366 (residue numbers in TcaPGK) (Fig. 2). By comparing the open and closed structures of apo TcaPGK and TmaPGK, respectively, we are able to determine the features essential for the conformational change of PGK. In the open conformation, an interdomain salt bridge between residues Arg148 in the N-domain and Glu375 in the C-domain forms a strap that ties the two domains together. In the closed conformation, Arg148 (Arg151 in TmaPGK) interacts with the phosphate group of 3-PG, not with Glu375 (Glu381 in TmaPGK); i.e., a change in the orientation of Arg148 enables it to switch from forming an interdomain salt bridge with Glu375 (open conformation) to forming a new intradomain salt bridge with the phosphate group of 3-PG bound (closed conformation). In addition, Arg60 in the N-domain (Arg62 in TmaPGK) forms a new interdomain salt bridge with Asp197 in the C-domain (Asp200 in TmaPGK), which stabilizes the closed conformation. By contrast, Arg60 participates in no direct interactions within the open structure of TcaPGK. Beyond the Ca the Arg60 residue shows little density within the structure of TcaPGK, which implies that Arg60 is intrinsically flexible in the absence of substrate (overall B factor val˚ 2, B factor value of Arg60 ue of TcaPGK structure: 32.9 A 2 ˚ ). Likewise, Asp197 also appears to particiresidue: 43.6 A pate in no direct interactions within the open structure of TcaPGK. Notably, the four residues involved in the salt bridge switch are strictly conserved in all known prokaryotic and eukaryotic PGK sequences (e.g., Fig. 1B). Moreover, it appears that it is reorientation of the interdomain salt bridges after substrate binding that brings the two domains into closer proximity (Fig. 3A and B). Analyses of the nine reported PGK structures revealed that Arg60-Asp197-like salt bridges are observed only
Fig. 2. Comparison of the open and closed conformations of PGK. Shown is a stereo view of the superposition of the structures of TcaPGK (open conformation: slate blue) and TmaPGK (closed conformation: orange) complexed with AMPPNP and 3-PG. Rotation (about 15.8°) of the C-domain may induce the closed conformation. TcaPGK residue numbers are shown in blue, while TmaPGK residue numbers are shown in orange. The figures were made using Pymol [19].
1048
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049
Fig. 3. Domain movement, including interdomain salt bridge switching. (A) Stereo view of the interdomain interactions within the structures of TcaPGK (open conformation: slate blue) and TmaPGK (closed conformation: orange). TcaPGK residue numbers are shown in blue, while TmaPGK residue numbers are shown in orange. (B) Electron density map (2Fo Fc map contoured at 1d) around the Arg148-Glu375 salt bridge within TcaPGK. (C) Salt bridge switching during PGK domain closure is depicted in a cartoon model.
within closed PGK structures (ternary complex: PDB code 1VPE and 13PK) [3,10], and that Arg148-Glu375-like salt bridges are observed within partially closed structures (PDB code 1PHP and 16PK) complexed with ADP or bisubstrate analogue [9,11]. This suggests that nucleotide binding alone is not sufficient to break the Arg148Glu375 salt bridge. On the other hand, 3-PG-complexed structures have no Arg148-Glu375-like salt bridges, although they still resemble the open conformation (PDB code 1HDI, 1KF0, and 3PGK) [5,7,8]. Most likely the binding of 3-PG disrupts the Arg148-Glu375 salt bridge by enabling formation of a new salt bridge between Arg148 and the phosphate group of 3-PG, which is observed in all known 3-PG-complexed structures. In summary, we have resolved the structure of TcaPGK in the absence of substrates (open conformation). Comparison of this open structure with a closed one (TmaPGK) revealed substrate-induced conformational changes, including interdomain salt bridge switching. The formation of an interdomain salt bridge between Arg148 and Glu375 stabilizes the open conformation. The binding of 3-PG disrupts that salt bridge and enables the formation of a new
interdomain salt bridge between Arg60 and Asp197, which stabilizes the closed conformation (Fig. 3C). Acknowledgments We thank Professor N. Sakabe and Drs. N. Igarashi and N. Matsugaki for their kind support during data collection at BL18B of the Photon Factory (Tsukuba, Japan). This research was supported by a grant from the KRIBB Research Initiative Program. References [1] R.D. Banks, C.C.F. Blake, P.R. Evans, R. Haser, D.W. Rice, G.W. Hardy, M. Merrett, A.W. Phillips, Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme, Nature 279 (1979) 773–777. [2] C.C. Blake, D.W. Rice, Phosphoglycerate kinase, Phil. Trans. Soc. Ser. A 293 (1981) 93–104. [3] B.E. Bernstein, P.A. Michels, W.G. Hol, Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation, Nature 385 (1997) 275–278.
J.H. Lee et al. / Biochemical and Biophysical Research Communications 350 (2006) 1044–1049 [4] Z. Kovari, M. Vas, Protein conformer selection by sequencedependent packing contacts in crystals of 3-phosphoglycerate kinase, Proteins 55 (2004) 198–209. ˚ resolution [5] A.N. Szilagyi, M. Ghosh, E. Garman, M. Vas, A 1.8 A structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and 3-phosphoglycerate in open conformation: new insight into the role of the nucleotide in domain closure, J. Mol. Biol. 306 (2001) 499–511. [6] B. Flachner, Z. Kovari, A. Varga, Z. Gugolya, F. Vonderviszt, G. Naray-Szabo, M. Vas, Role of phosphate chain mobility of MgATP in completing the 3-phosphoglycerate kinase catalytic site: binding, kinetic, and crystallographic studies with ATP and MgATP, Biochemistry 43 (2004) 3436–3449. [7] Z. Kovari, B. Flachner, G. Naray-Szabo, M. Vas, Crystallographic and thiol-reactivity studies on the complex of pig muscle phosphoglycerate kinase with ATP analogues: correlation between nucleotide binding mode and helix flexibility, Biochemistry 41 (2002) 8796–8806. [8] H.C. Watson, N.P. Walker, P.J. Shaw, T.N. Bryant, P.L. Wendell, L.A. Fothergill, R.E. Perkins, S.C. Conroy, M.J. Dobson, M.F. Tuite, A.J. Kingsman, S.M. Kingsman, Sequence and structure of yeast phosphoglycerate kinase, EMBO J. 1 (1982) 1635–1640. [9] G.J. Davies, S.J. Gamblin, J.A. Littlechild, Z. Dauter, K.S. Wilson, H.C. Watson, Structure of the ADP complex of the 3-phosphoglyc˚ , Acta Cryserate kinase from Bacillus stearothermophilus at 1.65 A tallogr. D Biol. Crystallogr. 50 (1994) 202–209. [10] G. Auerbach, R. Huber, M. Grattinger, K. Zaiss, H. Schurig, R. Jaenicke, U. Jacob, Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability, Structure 5 (1997) 1475–1483. [11] B.E. Bernstein, D.M. Williams, J.C. Bressi, P. Kuhn, M.H. Gelb, G.M. Blackburn, W.G. Hol, A bisubstrate analog induces unexpected
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
1049
conformational changes in phosphoglycerate kinase from Trypanosoma brucei, J. Mol. Biol. 279 (1998) 1137–1148. J.H. Lee, Y.J. Im, S-H. Rho, S.H. Park, G.B. Kang, S.J. Cho, T.-Y. Kim, H-J. Shin, D.-S. Lee, S.H. Eom, Crystallization and preliminary X-ray crystallographic studies of phosphoglycerate kinase from Thermus caldophilus, Mol. Cells 15 (2003) 370–372. A. Vagin, A. Teplyakov, MOLREP: an automated program for molecular replacement, J. Appl. Cryst. 30 (1997) 1022–1025. A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. GrosseKunstleve, J-S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography and NMR system: a new software suite for macromolecular structure determination, Acta Crystallogr. Sect. D Biol. Crystallogr. 54 (1998) 905–921. T.A. Jones, J.Y. Zhou, S.W. Cowan, M. Kjeldgaard, Improved methods for building protein models in electron density maps and the location of errors in these models, Acta Crystallogr. A 47 (1991) 110– 119. S. Hayward, H.J.C. Berendsen, Systematic analysis of domain motions in proteins from conformational change; new results on citrate synthase and T4 lysozyme, Proteins 30 (1998) 144– 154. R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK—a program to check the stereochemical quality of protein structures, J. Appl. Cryst. 26 (1993) 283–291. J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res. 24 (1997) 4876–4882. W.L. DeLano, The PyMOL User’s Manual, DeLano Scientific, San Carlos, CA, 2002.