Changes at the KinA PAS-A dimerization interface influence histidine kinase function

James Lee, Diana R Tomchick, Chad A Brautigam, Mischa Machius, Remco Kort, Klaas J Hellingwerf, Kevin H Gardner

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatory role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using beta-sheet/beta-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.

Original languageEnglish
Pages (from-to)4051-64
Number of pages14
JournalBiochemistry
Volume47
Issue number13
DOIs
Publication statusPublished - 1 Apr 2008

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Dimerization
Histidine
Dimers
Phosphotransferases
Ultracentrifugation
Site-Directed Mutagenesis
Gel Chromatography
Mutagenesis
Proteins
Magnetic Resonance Spectroscopy
X-Rays
Chromatography
Crystal lattices
Food
Protein Kinases
Nuclear magnetic resonance spectroscopy
Nutrients
Catalyst activity
Enzymes
Crystal structure

Keywords

  • Amino Acid Sequence
  • Bacillus subtilis
  • Bacterial Proteins
  • Crystallography, X-Ray
  • Dimerization
  • Histidine Kinase
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Kinases
  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

Cite this

Lee, J., Tomchick, D. R., Brautigam, C. A., Machius, M., Kort, R., Hellingwerf, K. J., & Gardner, K. H. (2008). Changes at the KinA PAS-A dimerization interface influence histidine kinase function. Biochemistry, 47(13), 4051-64. https://doi.org/10.1021/bi7021156
Lee, James ; Tomchick, Diana R ; Brautigam, Chad A ; Machius, Mischa ; Kort, Remco ; Hellingwerf, Klaas J ; Gardner, Kevin H. / Changes at the KinA PAS-A dimerization interface influence histidine kinase function. In: Biochemistry. 2008 ; Vol. 47, No. 13. pp. 4051-64.
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Lee, J, Tomchick, DR, Brautigam, CA, Machius, M, Kort, R, Hellingwerf, KJ & Gardner, KH 2008, 'Changes at the KinA PAS-A dimerization interface influence histidine kinase function' Biochemistry, vol. 47, no. 13, pp. 4051-64. https://doi.org/10.1021/bi7021156

Changes at the KinA PAS-A dimerization interface influence histidine kinase function. / Lee, James; Tomchick, Diana R; Brautigam, Chad A; Machius, Mischa; Kort, Remco; Hellingwerf, Klaas J; Gardner, Kevin H.

In: Biochemistry, Vol. 47, No. 13, 01.04.2008, p. 4051-64.

Research output: Contribution to JournalArticleAcademicpeer-review

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T1 - Changes at the KinA PAS-A dimerization interface influence histidine kinase function

AU - Lee, James

AU - Tomchick, Diana R

AU - Brautigam, Chad A

AU - Machius, Mischa

AU - Kort, Remco

AU - Hellingwerf, Klaas J

AU - Gardner, Kevin H

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AB - The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatory role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using beta-sheet/beta-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.

KW - Amino Acid Sequence

KW - Bacillus subtilis

KW - Bacterial Proteins

KW - Crystallography, X-Ray

KW - Dimerization

KW - Histidine Kinase

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Conformation

KW - Protein Kinases

KW - Journal Article

KW - Research Support, N.I.H., Extramural

KW - Research Support, Non-U.S. Gov't

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VL - 47

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EP - 4064

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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ER -

Lee J, Tomchick DR, Brautigam CA, Machius M, Kort R, Hellingwerf KJ et al. Changes at the KinA PAS-A dimerization interface influence histidine kinase function. Biochemistry. 2008 Apr 1;47(13):4051-64. https://doi.org/10.1021/bi7021156