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Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: A model for changes in dynamics upon target binding

Åkerud, Tomas LU ; Thulin, Eva LU ; Van Etten, RL and Akke, Mikael LU (2002) In Journal of Molecular Biology 322(1). p.137-152
Abstract
Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K-d = 1.5(+/-0.1) mM and an off-rate on the order of 10(4) s(-1).H-1 and N-15 NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. N-15 relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory... (More)
Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K-d = 1.5(+/-0.1) mM and an off-rate on the order of 10(4) s(-1).H-1 and N-15 NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. N-15 relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer. (C) 2002 Elsevier Science Ltd. All rights reserved. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
parameters, order, enzyme dynamics, conformational exchange, dimerization, rotational diffusion tensor
in
Journal of Molecular Biology
volume
322
issue
1
pages
137 - 152
publisher
Elsevier
external identifiers
  • wos:000178205100012
  • pmid:12215420
  • scopus:0036967268
ISSN
1089-8638
DOI
10.1016/S0022-2836(02)00714-3
language
English
LU publication?
yes
id
24dab903-f143-474e-82ca-0d0049c68289 (old id 326396)
date added to LUP
2007-08-06 13:53:45
date last changed
2017-11-12 04:01:57
@article{24dab903-f143-474e-82ca-0d0049c68289,
  abstract     = {Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K-d = 1.5(+/-0.1) mM and an off-rate on the order of 10(4) s(-1).H-1 and N-15 NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. N-15 relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer. (C) 2002 Elsevier Science Ltd. All rights reserved.},
  author       = {Åkerud, Tomas and Thulin, Eva and Van Etten, RL and Akke, Mikael},
  issn         = {1089-8638},
  keyword      = {parameters,order,enzyme dynamics,conformational exchange,dimerization,rotational diffusion tensor},
  language     = {eng},
  number       = {1},
  pages        = {137--152},
  publisher    = {Elsevier},
  series       = {Journal of Molecular Biology},
  title        = {Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: A model for changes in dynamics upon target binding},
  url          = {http://dx.doi.org/10.1016/S0022-2836(02)00714-3},
  volume       = {322},
  year         = {2002},
}