Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: Relationship between substrate antagonism and interdomain communication

A. Merli, A. N. Szilágyi, B. Flachner, G. L. Rossi, M. Vas

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, β,γ-methyleneadenosine-5′-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2′3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the Kd values for MgADP were found to be 0.096 ± 0.021 and 0.045 ± 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both Kd values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 ± 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 ± 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg2+-free) binding (Kd = 0.34 ± 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg2+ ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg2+ binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.

Original languageEnglish
Pages (from-to)111-119
Number of pages9
JournalBiochemistry
Volume41
Issue number1
DOIs
Publication statusPublished - Jan 8 2002

Fingerprint

Phosphoglycerate Kinase
Muscle
Swine
Nucleotides
Communication
Muscles
Adenosine Diphosphate
Crystals
Substrates
Conformations
Adenosine Triphosphate
3-phosphoglycerate
Titration
Crystal lattices
Arginine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution : Relationship between substrate antagonism and interdomain communication. / Merli, A.; Szilágyi, A. N.; Flachner, B.; Rossi, G. L.; Vas, M.

In: Biochemistry, Vol. 41, No. 1, 08.01.2002, p. 111-119.

Research output: Contribution to journalArticle

@article{075c398d5ca14dea9bcfb10b7be98054,
title = "Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: Relationship between substrate antagonism and interdomain communication",
abstract = "Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, β,γ-methyleneadenosine-5′-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2′3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the Kd values for MgADP were found to be 0.096 ± 0.021 and 0.045 ± 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both Kd values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 ± 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 ± 0.01 mM). Thus, the {"}substrate antagonism{"} observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg2+-free) binding (Kd = 0.34 ± 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg2+ ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg2+ binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.",
author = "A. Merli and Szil{\'a}gyi, {A. N.} and B. Flachner and Rossi, {G. L.} and M. Vas",
year = "2002",
month = "1",
day = "8",
doi = "10.1021/bi0115380",
language = "English",
volume = "41",
pages = "111--119",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution

T2 - Relationship between substrate antagonism and interdomain communication

AU - Merli, A.

AU - Szilágyi, A. N.

AU - Flachner, B.

AU - Rossi, G. L.

AU - Vas, M.

PY - 2002/1/8

Y1 - 2002/1/8

N2 - Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, β,γ-methyleneadenosine-5′-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2′3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the Kd values for MgADP were found to be 0.096 ± 0.021 and 0.045 ± 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both Kd values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 ± 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 ± 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg2+-free) binding (Kd = 0.34 ± 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg2+ ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg2+ binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.

AB - Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, β,γ-methyleneadenosine-5′-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2′3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the Kd values for MgADP were found to be 0.096 ± 0.021 and 0.045 ± 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both Kd values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 ± 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 ± 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg2+-free) binding (Kd = 0.34 ± 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg2+ ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg2+ binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.

UR - http://www.scopus.com/inward/record.url?scp=0037039416&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037039416&partnerID=8YFLogxK

U2 - 10.1021/bi0115380

DO - 10.1021/bi0115380

M3 - Article

C2 - 11772008

AN - SCOPUS:0037039416

VL - 41

SP - 111

EP - 119

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 1

ER -