Nucleotide-induced conformational transitions in the CBS domain protein MJ0729 of Methanocaldococcus jannaschii.
Nucleotide-binding cystathionine β-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 µM, 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.
Martínez-Cruz
Luis Alfonso
LA
Unidad de Biología Estructural, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio (Vizcaya), Spain. amartinez@cicbiogune.es
Encinar
José Antonio
JA
Sevilla
Paz
P
Oyenarte
Iker
I
Gómez-García
Inmaculada
I
Aguado-Llera
David
D
García-Blanco
Francisco
F
Gómez
Javier
J
Neira
José L
JL
D000241:Adenosine / Q000031:analogs & derivatives*; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D017016:Methanococcaceae / Q000737:chemistry / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared
D009711:Nucleotides; D003541:Cystathionine beta-Synthase; D000241:Adenosine