scholarly journals Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites

2004 ◽  
Vol 380 (3) ◽  
pp. 805-813 ◽  
Author(s):  
Michael A. CATER ◽  
John FORBES ◽  
Sharon La FONTAINE ◽  
Diane COX ◽  
Julian F. B. MERCER
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Intracellular Trafficking ◽  
Copper Toxicity ◽  
Site Directed Mutagenesis ◽  
Metal Binding Sites ◽  
Wilson Protein ◽  
Necessary And Sufficient ◽  
Golgi Network

The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4–6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1–5 (amino acids 64–540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.

scholarly journals Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)

2006 ◽  
Vol 401 (1) ◽  
pp. 143-153 ◽  
Author(s):  
Michael A. Cater ◽  
Sharon La fontaine ◽  
Julian F. B. Mercer
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Copper Binding ◽  
Phosphatase Domain ◽  
Copper Chelation ◽  
Metal Binding Sites ◽  
Excess Copper ◽  
Wilson Protein ◽  
P Type

The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hep-atocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular traf-ficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mut-ation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the consti-tutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites

Biochemistry ◽  
1992 ◽  
Vol 31 (24) ◽  
pp. 5449-5458 ◽  
Author(s):  
John Jenkins ◽  
Joel Janin ◽  
Felix Rey ◽  
Mohammed Chiadmi ◽  
Herman Van Tilbeurgh ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Metal Binding ◽  
Binding Sites ◽  
Glucose Isomerase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Metal Binding Sites

scholarly journals Crystal Structures of Apo and Metal-Bound Forms of the UreE Protein fromHelicobacter pylori: Role of Multiple Metal Binding Sites,

Biochemistry ◽  
2010 ◽  
Vol 49 (33) ◽  
pp. 7080-7088 ◽  
Author(s):  
Rong Shi ◽  
Christine Munger ◽  
Abdalin Asinas ◽  
Stéphane L. Benoit ◽  
Erica Miller ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Binding Sites

scholarly journals The Role of GMXCXXC Metal Binding Sites in the Copper-induced Redistribution of the Menkes Protein

1999 ◽  
Vol 274 (16) ◽  
pp. 11170-11177 ◽  
Author(s):  
Daniel Strausak ◽  
Sharon La Fontaine ◽  
Joanne Hill ◽  
Stephen D. Firth ◽  
Paul J. Lockhart ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Metal Binding Sites ◽  
Menkes Protein

scholarly journals BioMetAll: Identifying Metal-Binding Sites in Proteins from Backbone Preorganization

2020 ◽  
Author(s):  
José-Emilio Sánchez-Aparicio ◽  
Laura Tiessler-Sala ◽  
Lorea Velasco-Carneros ◽  
Lorena Roldán-Martín ◽  
Giuseppe Sciortino ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
De Novo ◽  
Conformational Transition ◽  
Protein Backbone ◽  
Metal Binding Sites ◽  
Structural Match ◽  
Geometric Descriptors ◽  
Metal Migration

<div><div><div><p>With a large amount of research dedicated to decoding how metallic species bind to protein, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parametrization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 50 metal-binding X-Ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems which structures (either experimental or theoretical) are not optimal for metal binding sites. We report here its application on three different challenging cases i) the modulation of metal-binding sites during conformational transition in human serum albumin, ii) the identification of possible routes of metal migration in hemocyanins, and iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology, and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding.</p></div></div></div>

scholarly journals Escherichia coli inorganic pyrophosphatase : site-directed mutagenesis of the metal binding sites

FEBS Letters ◽  
1996 ◽  
Vol 399 (1-2) ◽  
pp. 99-102 ◽  
Author(s):  
Svetlana Avaeva ◽  
Pavel Ignatov ◽  
Svetlana Kurilova ◽  
Tatjana Nazarova ◽  
Elena Rodina ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Inorganic Pyrophosphatase ◽  
Directed Mutagenesis ◽  
Metal Binding Sites

scholarly journals BioMetAll: Identifying Metal-Binding Sites in Proteins from Backbone Preorganization

2020 ◽  
Author(s):  
José-Emilio Sánchez-Aparicio ◽  
Laura Tiessler-Sala ◽  
Lorea Velasco-Carneros ◽  
Lorena Roldán-Martín ◽  
Giuseppe Sciortino ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
De Novo ◽  
Conformational Transition ◽  
Protein Backbone ◽  
Metal Binding Sites ◽  
Structural Match ◽  
Geometric Descriptors ◽  
Metal Migration

<div><div><div><p>With a large amount of research dedicated to decoding how metallic species bind to protein, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parametrization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 50 metal-binding X-Ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems which structures (either experimental or theoretical) are not optimal for metal binding sites. We report here its application on three different challenging cases i) the modulation of metal-binding sites during conformational transition in human serum albumin, ii) the identification of possible routes of metal migration in hemocyanins, and iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology, and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding.</p></div></div></div>

scholarly journals Rational design of metal-binding sites in domain-swapped myoglobin dimers

2021 ◽  
Vol 217 ◽  
pp. 111374
Author(s):  
Satoshi Nagao ◽  
Ayaka Idomoto ◽  
Naoki Shibata ◽  
Yoshiki Higuchi ◽  
Shun Hirota
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Rational Design ◽  
Metal Binding Sites
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