3 research outputs found
EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the NāTerminal Domain of the Copper Transporter, Ctr1
Copper is an essential metal whose
localization within the cells
must be carefully controlled to avoid copper dependent redox cycling.
Although most of the key proteins involved in cellular copper transfer
have been identified, fundamental questions regarding the copper transfer
mechanism have yet to be resolved. One of the blood carrier proteins
believed to be involved in copper transfer to the cell is human serum
albumin (HSA). However, direct evidence for close interaction between
HSA and the extracellular domain of the copper transporter Ctr1 has
not yet been found. By utilizing EPR spectroscopy, we show here that
HSA closely interacts with the first 14 amino acids of the Ctr1, even
without the presence of copper ions
Insights into the N-terminal Cu(II) and Cu(I) binding sites of the human copper transporter CTR1
<p>Copper transporter 1 (CTR1) is the main copper transporter in the eukaryotic system. CTR1 has several important roles: It binds Cu(II) ions that are present in the blood; it reduces those Cu(II) ions to Cu(I); and it subsequently transfers Cu(I) to the cytoplasmic domain, where the ion is delivered to various cellular pathways. Here, we seek to identify CTR1 binding sites for Cu(II) and Cu(I) and to shed light on the Cu(II)-to-Cu(I) reduction process. We focus on the first 14 amino acids of CTR1. This N-terminal segment is rich with histidine and methionine residues, which are known to bind Cu(II) and Cu(I), respectively; thus, this region has been suggested to have an important function in recruiting Cu(II) and reducing it to Cu(I). We utilize electron paramagnetic resonance (EPR) spectroscopy together with nuclear magnetic resonance (NMR) and UV-VIS spectroscopy and alanine substitution to reveal Cu(II) and Cu(I) binding sites in the focal 14-amino-acid segment. We show that H5 and H6 directly coordinate to Cu(II), whereas M7, M9, and M12 are involved in Cu(I) binding. This research is another step on the way to a complete understanding of the cellular copper regulation mechanism in humans.</p
Revealing the DNA Binding Modes of CsoR by EPR Spectroscopy
In pathogens, a unique class of metalloregulator proteins,
called
gene regulatory proteins, sense specific metal ions that initiate
gene transcription of proteins that export metal ions from the cell,
thereby preventing toxicity and cell death. CsoR is a metalloregulator
protein found in various bacterial systems that āsenseā
Cu(I) ions with high affinity. Upon copper binding, CsoR dissociates
from the DNA promoter region, resulting in initiation of gene transcription.
Crystal structures of CsoR in the presence and absence of Cu(I) from
various bacterial systems have been reported, suggesting either a
dimeric or tetrameric structure of these helical proteins. However,
structural information about the CsoR-DNA complex is missing. Here,
we applied electron paramagnetic resonance (EPR) spectroscopy to follow
the conformational and dynamical changes that Mycobacterium
tuberculosis CsoR undergoes upon DNA binding in solution.
We showed that the quaternary structure is predominantly dimeric in
solution, and only minor conformational and dynamical changes occur
in the DNA bound state. Also, labeling of the unresolved C- terminus
revealed no significant change in dynamics upon DNA binding. These
observations are unique, since for other bacterial copper metalloregulators,
such as the MerR and CopY families, major conformational changes were
observed upon DNA binding, indicating a different mode of action for
this protein family