Insights into the Molybdenum/Copper Heterometallic Cluster Assembly in the Orange Protein

Probing Intermolecular Interactions with an Artificial Metal-Binding ATCUN Tag

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [S2MoVIS2CuIS2MoVIS2]3- (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of CuII plus tetrathiomolybdate to apo-ORP under controlled conditions. In the work described herein, we artificially inserted the ATCUN ("amino terminus Cu and Ni") motif in the Desulfovibrio gigas ORP (Ala1Ser2His3 followed by the native amino acid residues; modified protein abbreviated as ORP∗) to increase our understanding of the Mo/Cu cluster assembly in ORP. The apo-ORP∗ binds CuII in a 1:1 ratio to yield CuII-ORP∗, as clearly demonstrated by EPR (g||,⊥ = 2.183, 2.042 and ACu ||,⊥ = 207 × 10-4 cm-1, 19 × 10-4 cm-1) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ϵ = 90 M-1 cm-1). The 1H NMR spectrum shows that His3 and His53 are significantly affected upon the addition of the CuII. The X-ray structure shows that these two residues are very far apart (Cα-Cα ≈ 27.9 Å), leading us to suggest that the metal-induced NMR perturbations are due to the interaction of two protein molecules with a single metal ion. Docking analysis supports the metal-mediated dimer formation. The subsequent tetrathiomolybdate binding, to yield the native Mo/Cu cluster, occurs only upon addition of dithiothreitol, as shown by UV-visible and NMR spectroscopies. Additionally, 1H NMR of AgI-ORP∗ (AgI used as a surrogate of CuI) showed that AgI strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that CuII and CuI have two different binding sites in ORP∗. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that CuII is reduced to CuI and transferred from the ATCUN motif to the methionine site; finally, CuI is transferred to the cluster-binding region, upon the interaction of two protein molecules. This result may suggest that copper trafficking is triggered by redox-dependent coordination properties of copper in a trafficking pathway.

Original languageEnglish
Pages (from-to)8900-8911
Number of pages12
JournalInorganic Chemistry
Volume56
Issue number15
DOIs
Publication statusPublished - 7 Aug 2017

Fingerprint

Molybdenum
molybdenum
Copper
assembly
Metals
proteins
copper
metals
Proteins
interactions
methionine
nuclear magnetic resonance
Nuclear magnetic resonance
Methionine
Molecules
Bacterial Proteins
Dithiothreitol
Sulfur
Dimers
Nuclear magnetic resonance spectroscopy

Keywords

  • DESULFOVIBRIO-VULGARIS HILDENBOROUGH
  • MO-CU CLUSTER
  • WILSONS-DISEASE
  • COPPER PROTEINS
  • STRUCTURAL-CHARACTERIZATION
  • H-1-NMR SPECTROSCOPY
  • SECONDARY STRUCTURE
  • SERUM-ALBUMIN
  • PRION PROTEIN
  • ION COMPLEXES

Cite this

@article{4c85c47cacbd49a0b8b2b7bc9c633931,
title = "Insights into the Molybdenum/Copper Heterometallic Cluster Assembly in the Orange Protein: Probing Intermolecular Interactions with an Artificial Metal-Binding ATCUN Tag",
abstract = "Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [S2MoVIS2CuIS2MoVIS2]3- (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of CuII plus tetrathiomolybdate to apo-ORP under controlled conditions. In the work described herein, we artificially inserted the ATCUN ({"}amino terminus Cu and Ni{"}) motif in the Desulfovibrio gigas ORP (Ala1Ser2His3 followed by the native amino acid residues; modified protein abbreviated as ORP∗) to increase our understanding of the Mo/Cu cluster assembly in ORP. The apo-ORP∗ binds CuII in a 1:1 ratio to yield CuII-ORP∗, as clearly demonstrated by EPR (g||,⊥ = 2.183, 2.042 and ACu ||,⊥ = 207 × 10-4 cm-1, 19 × 10-4 cm-1) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ϵ = 90 M-1 cm-1). The 1H NMR spectrum shows that His3 and His53 are significantly affected upon the addition of the CuII. The X-ray structure shows that these two residues are very far apart (Cα-Cα ≈ 27.9 {\AA}), leading us to suggest that the metal-induced NMR perturbations are due to the interaction of two protein molecules with a single metal ion. Docking analysis supports the metal-mediated dimer formation. The subsequent tetrathiomolybdate binding, to yield the native Mo/Cu cluster, occurs only upon addition of dithiothreitol, as shown by UV-visible and NMR spectroscopies. Additionally, 1H NMR of AgI-ORP∗ (AgI used as a surrogate of CuI) showed that AgI strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that CuII and CuI have two different binding sites in ORP∗. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that CuII is reduced to CuI and transferred from the ATCUN motif to the methionine site; finally, CuI is transferred to the cluster-binding region, upon the interaction of two protein molecules. This result may suggest that copper trafficking is triggered by redox-dependent coordination properties of copper in a trafficking pathway.",
keywords = "DESULFOVIBRIO-VULGARIS HILDENBOROUGH, MO-CU CLUSTER, WILSONS-DISEASE, COPPER PROTEINS, STRUCTURAL-CHARACTERIZATION, H-1-NMR SPECTROSCOPY, SECONDARY STRUCTURE, SERUM-ALBUMIN, PRION PROTEIN, ION COMPLEXES",
author = "Maiti, {Biplab K.} and Almeida, {Rui M.} and Maia, {Luisa B.} and Isabel Moura and Moura, {Jos{\'e} J.G.}",
note = "sem pdf conforme despacho. FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). B.K.M., L.B.M, and RM.A. thank FCT/MEC for the fellowship grants (SFRH/BPD/63066/2009, SFRH/BPD/111404/2015, and SFRH/BPD/80293/2011, respectively), which are financed by national funds and co-financed by FSE. The NMR spectrometers are part of the National NMR Network (RNRMN) and are funded by FCT/MEC (project RECI/BBB-BQB/0230/2012).",
year = "2017",
month = "8",
day = "7",
doi = "10.1021/acs.inorgchem.7b00840",
language = "English",
volume = "56",
pages = "8900--8911",
journal = "Inorganic Chemistry",
issn = "0020-1669",
publisher = "American Chemical Society",
number = "15",

}

TY - JOUR

T1 - Insights into the Molybdenum/Copper Heterometallic Cluster Assembly in the Orange Protein

T2 - Probing Intermolecular Interactions with an Artificial Metal-Binding ATCUN Tag

AU - Maiti, Biplab K.

AU - Almeida, Rui M.

AU - Maia, Luisa B.

AU - Moura, Isabel

AU - Moura, José J.G.

N1 - sem pdf conforme despacho. FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). B.K.M., L.B.M, and RM.A. thank FCT/MEC for the fellowship grants (SFRH/BPD/63066/2009, SFRH/BPD/111404/2015, and SFRH/BPD/80293/2011, respectively), which are financed by national funds and co-financed by FSE. The NMR spectrometers are part of the National NMR Network (RNRMN) and are funded by FCT/MEC (project RECI/BBB-BQB/0230/2012).

PY - 2017/8/7

Y1 - 2017/8/7

N2 - Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [S2MoVIS2CuIS2MoVIS2]3- (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of CuII plus tetrathiomolybdate to apo-ORP under controlled conditions. In the work described herein, we artificially inserted the ATCUN ("amino terminus Cu and Ni") motif in the Desulfovibrio gigas ORP (Ala1Ser2His3 followed by the native amino acid residues; modified protein abbreviated as ORP∗) to increase our understanding of the Mo/Cu cluster assembly in ORP. The apo-ORP∗ binds CuII in a 1:1 ratio to yield CuII-ORP∗, as clearly demonstrated by EPR (g||,⊥ = 2.183, 2.042 and ACu ||,⊥ = 207 × 10-4 cm-1, 19 × 10-4 cm-1) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ϵ = 90 M-1 cm-1). The 1H NMR spectrum shows that His3 and His53 are significantly affected upon the addition of the CuII. The X-ray structure shows that these two residues are very far apart (Cα-Cα ≈ 27.9 Å), leading us to suggest that the metal-induced NMR perturbations are due to the interaction of two protein molecules with a single metal ion. Docking analysis supports the metal-mediated dimer formation. The subsequent tetrathiomolybdate binding, to yield the native Mo/Cu cluster, occurs only upon addition of dithiothreitol, as shown by UV-visible and NMR spectroscopies. Additionally, 1H NMR of AgI-ORP∗ (AgI used as a surrogate of CuI) showed that AgI strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that CuII and CuI have two different binding sites in ORP∗. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that CuII is reduced to CuI and transferred from the ATCUN motif to the methionine site; finally, CuI is transferred to the cluster-binding region, upon the interaction of two protein molecules. This result may suggest that copper trafficking is triggered by redox-dependent coordination properties of copper in a trafficking pathway.

AB - Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [S2MoVIS2CuIS2MoVIS2]3- (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of CuII plus tetrathiomolybdate to apo-ORP under controlled conditions. In the work described herein, we artificially inserted the ATCUN ("amino terminus Cu and Ni") motif in the Desulfovibrio gigas ORP (Ala1Ser2His3 followed by the native amino acid residues; modified protein abbreviated as ORP∗) to increase our understanding of the Mo/Cu cluster assembly in ORP. The apo-ORP∗ binds CuII in a 1:1 ratio to yield CuII-ORP∗, as clearly demonstrated by EPR (g||,⊥ = 2.183, 2.042 and ACu ||,⊥ = 207 × 10-4 cm-1, 19 × 10-4 cm-1) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ϵ = 90 M-1 cm-1). The 1H NMR spectrum shows that His3 and His53 are significantly affected upon the addition of the CuII. The X-ray structure shows that these two residues are very far apart (Cα-Cα ≈ 27.9 Å), leading us to suggest that the metal-induced NMR perturbations are due to the interaction of two protein molecules with a single metal ion. Docking analysis supports the metal-mediated dimer formation. The subsequent tetrathiomolybdate binding, to yield the native Mo/Cu cluster, occurs only upon addition of dithiothreitol, as shown by UV-visible and NMR spectroscopies. Additionally, 1H NMR of AgI-ORP∗ (AgI used as a surrogate of CuI) showed that AgI strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that CuII and CuI have two different binding sites in ORP∗. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that CuII is reduced to CuI and transferred from the ATCUN motif to the methionine site; finally, CuI is transferred to the cluster-binding region, upon the interaction of two protein molecules. This result may suggest that copper trafficking is triggered by redox-dependent coordination properties of copper in a trafficking pathway.

KW - DESULFOVIBRIO-VULGARIS HILDENBOROUGH

KW - MO-CU CLUSTER

KW - WILSONS-DISEASE

KW - COPPER PROTEINS

KW - STRUCTURAL-CHARACTERIZATION

KW - H-1-NMR SPECTROSCOPY

KW - SECONDARY STRUCTURE

KW - SERUM-ALBUMIN

KW - PRION PROTEIN

KW - ION COMPLEXES

UR - http://www.scopus.com/inward/record.url?scp=85027003124&partnerID=8YFLogxK

U2 - 10.1021/acs.inorgchem.7b00840

DO - 10.1021/acs.inorgchem.7b00840

M3 - Article

VL - 56

SP - 8900

EP - 8911

JO - Inorganic Chemistry

JF - Inorganic Chemistry

SN - 0020-1669

IS - 15

ER -