Insights into the Molybdenum/Copper Heterometallic Cluster Assembly in the Orange Protein: Probing Intermolecular Interactions with an Artificial Metal-Binding ATCUN Tag

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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

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

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