Stability and binding of the phosphorylated species of the N-terminal domain of enzyme I and the histidine phosphocarrier protein from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system

The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. It is formed by two general proteins: enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI is formed by two domains, with the N-terminal domain (EIN) being responsible for the binding to HPr. In low-G + C Gram-positive bacteria, HPr becomes phosphorylated not only by phosphoenolpyruvate (PEP) at the active-site histidine, but also by ATP at a serine. In this work, we have characterized: (i) the stability and binding affinities between the active-site-histidine phosphorylated species of HPr and the EIN from Streptomyces coelicolor; and (ii) the stability and binding affinities of the species involving the phosphorylation at the regulatory serine of HPrsc. Our results show that the phosphorylated active-site species of both proteins are less stable than the unphosphorylated counterparts. Conversely, the Hpr-S47D, which mimics phosphorylation at the regulatory serine, is more stable than wild-type HPrsc due to helical N-capping effects, as suggested by the modeled structure of the protein. Binding among the phosphorylated and unphosphorylated species is always entropically driven, but the affinity and the enthalpy vary widely

Deciphering the Binding between Nupr1 and MSL1 and Their DNA-Repairing Activity.

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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