Nucleotide-induced conformational transitions in the CBS domain protein MJ0729 of Methanocaldococcus jannaschii

Nucleotide-binding cystathionine -synthase (CBS) domains function as regulatory motifs in several proteins distributed through all kingdoms of life. This function has been proposed based on their affinity for adenosyl- derivatives, although the exact binding mechanisms remain largely unknown. The question of how CBS domains exactly work is relevant because in humans, several genetic diseases have been associated with mutations in those motifs. In this work, we describe the adenosyl-ligand (AMP, ATP, NADP and SAM) properties of the wild-type CBS domain protein MJ0729 from Methanocaldococcus jannaschii by using a combination of spectroscopic techniques (fluorescence, FTIR and FRET). The fluorescence results show that binding to AMP and ATP occurs with an apparent dissociation constant of ∼10 M, and interestingly enough, binding induces protein conformational changes, as shown by FTIR. On the other hand, fluorescence spectra (FRET and steady-state) did not change upon addition of NADP and SAM to MJ0729, suggesting that tryptophan and/or tyrosine residues were not involved in the recognition of those ligands; however, there were changes in the secondary structure of the protein upon addition of NADP and SAM, as shown by FTIR (thus, indicating binding to the nucleotide). Taken together, these results suggest that: (i) the adenosyl ligands bind to MJ0729 in different ways, and (ii) there are changes in the protein secondary structure upon binding of the nucleotides. The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals. permissionsoup.com2010 © The Author 2010. Published by Oxford University Press. All rights reserved.The work of LAMC work has been supported by grants of Departamento de Educacio´n, Universidades e Investigacio´n del Gobierno Vasco (PI2010-17), the Basque Government (ETORTEK IE05-147, IE07-202), Diputacio´n Foral de Bizkaia (Exp. 7/13/08/2006/11 and 7/13/08/2005/14), Spanish Ministerio de Ciencia e Innovacio´n (MICINN) (SAF2005-00855) and the MICINN CONSOLIDERINGENIO 2010 Program (CSD2008-00005). The work of J.A.E. has been supported by MICINN [BFU2008-00602] and the MICINN CONSOLIDER-INGENIO 2010 Program [CSD2008-00005]. The work of P.S.-S. and F.G.-B. has been supported by a grant from the Universidad Complutense de Madrid [Exp: 950247]. The work of J.G. has been supported by MICINN [SAF2008-05742-C02-01] and the Generalitat Valenciana [ACOMP/2010/114]. And finally, the work of J.L.N. has been supported by MICINN [SAF2008-05742- C02-01], the MICINN CONSOLIDER-INGENIO 2010 Program [CSD2008-00005], the Generalitat Valenciana [ACOMP/2010/114] and the FIPSE Foundation [Exp: 36557/ 06].Peer Reviewe

Nucleotide-induced conformational transitions in the CBS domain protein MJ0729 of Methanocaldococcus jannaschii

Nucleotide-binding cystathionine -synthase (CBS) domains function as regulatory motifs in several proteins distributed through all kingdoms of life. This function has been proposed based on their affinity for adenosyl- derivatives, although the exact binding mechanisms remain largely unknown. The question of how CBS domains exactly work is relevant because in humans, several genetic diseases have been associated with mutations in those motifs. In this work, we describe the adenosyl-ligand (AMP, ATP, NADP and SAM) properties of the wild-type CBS domain protein MJ0729 from Methanocaldococcus jannaschii by using a combination of spectroscopic techniques (fluorescence, FTIR and FRET). The fluorescence results show that binding to AMP and ATP occurs with an apparent dissociation constant of ∼10 M, and interestingly enough, binding induces protein conformational changes, as shown by FTIR. On the other hand, fluorescence spectra (FRET and steady-state) did not change upon addition of NADP and SAM to MJ0729, suggesting that tryptophan and/or tyrosine residues were not involved in the recognition of those ligands; however, there were changes in the secondary structure of the protein upon addition of NADP and SAM, as shown by FTIR (thus, indicating binding to the nucleotide). Taken together, these results suggest that: (i) the adenosyl ligands bind to MJ0729 in different ways, and (ii) there are changes in the protein secondary structure upon binding of the nucleotides. The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals. permissionsoup.com2010 © The Author 2010. Published by Oxford University Press. All rights reserved.The work of LAMC work has been supported by grants of Departamento de Educacio´n, Universidades e Investigacio´n del Gobierno Vasco (PI2010-17), the Basque Government (ETORTEK IE05-147, IE07-202), Diputacio´n Foral de Bizkaia (Exp. 7/13/08/2006/11 and 7/13/08/2005/14), Spanish Ministerio de Ciencia e Innovacio´n (MICINN) (SAF2005-00855) and the MICINN CONSOLIDERINGENIO 2010 Program (CSD2008-00005). The work of J.A.E. has been supported by MICINN [BFU2008-00602] and the MICINN CONSOLIDER-INGENIO 2010 Program [CSD2008-00005]. The work of P.S.-S. and F.G.-B. has been supported by a grant from the Universidad Complutense de Madrid [Exp: 950247]. The work of J.G. has been supported by MICINN [SAF2008-05742-C02-01] and the Generalitat Valenciana [ACOMP/2010/114]. And finally, the work of J.L.N. has been supported by MICINN [SAF2008-05742- C02-01], the MICINN CONSOLIDER-INGENIO 2010 Program [CSD2008-00005], the Generalitat Valenciana [ACOMP/2010/114] and the FIPSE Foundation [Exp: 36557/ 06].Peer Reviewe