Stalking metal-linked dimers

Pazehoski, Kristina O.; Collins, Tyler; Boyle, Robert; Jensen‐Seaman, Michael I.; Dameron, Charles T.

doi:10.1016/j.jinorgbio.2007.10.027

Cited by 7 publications

(5 citation statements)

References 64 publications

(86 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This cleavage impedes dimerization and dramatically lowers the DNA-binding affinity. Activation of CopR presumably proceeds by monomerization of the repressor dimer, as is the case for CopY of E. hirae [20,42]. The extended C-terminus of CopR features the metal-binding motif CXCX 6 CXC.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The copper-responsive repressor CopR of Lactococcus lactis is a ‘winged helix’ protein

Cantini

Banci

Solioz

2008

Biochemical Journal

View full text Add to dashboard Cite

CopR of Lactococcus lactis is a copper-responsive repressor involved in copper homoeostasis. It controls the expression of a total of 11 genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N-terminal domain to a 'cop box' of consensus TACANNTGTA, which is conserved in Firmicutes. We have solved the NMR solution structure of the N-terminal DNA-binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors, and the present structure represents a novel family of copper regulators, which we propose to call the CopY family.

show abstract

Section: Discussionmentioning

confidence: 99%

“…The Zn 2+ is bound to the CXCX 6 CXC metal-binding motif. Upon induction, two Cu + replaced the Zn 2+ and destabilized the repressor dimer, thereby lowering the affinity for DNA [19,20,42,43]. Copper is co-ordinated in a very stable solvent-shielded Cu + 2 -S 4 binuclear cluster [20].…”

Section: Discussionmentioning

confidence: 99%

The copper-responsive repressor CopR of Lactococcus lactis is a ‘winged helix’ protein

Cantini

Banci

Solioz

2008

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Previous ESI mass spectroscopy experiments provide additional support for this finding [25]. The ESI mass spectrum of GB1-Ymbs38 under soft ionization conditions [26] exhibits ions from which a dimeric molecular mass of 25.3 kDa can be derived, in addition to monomeric ions with a derived molecular mass of 12.6 kDa.…”

Section: Discussionmentioning

confidence: 71%

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

Pazehoski

Cobine

Winzor

et al. 2011

Biochemical and Biophysical Research Communications

Self Cite

View full text Add to dashboard Cite

Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin binding domain of Streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

show abstract

“…Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) is a highly accurate method to determine the quaternary structure of complexes of proteins compared to SEC [33]. A nanoelectrospray ionization time-of-flight (nESI-TOF) analyzer was used for this mass spectrometric analysis of rHSP-tag under native conditions.…”

Section: Resultsmentioning

confidence: 99%

16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

et al. 2013

View full text Add to dashboard Cite

BackgroundThe 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored.Methodology/Principal FindingsTo identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates.Conclusions/SignificanceThe concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.

show abstract

Stalking metal-linked dimers

Cited by 7 publications

References 64 publications

The copper-responsive repressor CopR of Lactococcus lactis is a ‘winged helix’ protein

The copper-responsive repressor CopR of Lactococcus lactis is a ‘winged helix’ protein

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

Contact Info

Product

Resources

About