Cu(I) recognition via cation-pi and methionine interactions in CusF.

Methionine-rich motifs have an important role in copper trafficking factors, including the CusF protein. Here we show that CusF uses a new metal recognition site wherein Cu(I) is tetragonally displaced from a Met2His ligand plane toward a conserved tryptophan. Spectroscopic studies demonstrate that both thioether ligation and strong cation-pi interactions with tryptophan stabilize metal binding. This novel active site chemistry affords mechanisms for control of adventitious metal redox and substitution chemistry.

A multinuclear copper(I) cluster forms the dimerization interface in copper-loaded human copper chaperone for superoxide dismutase.

Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.

Cysteine-to-serine mutants of the human copper chaperone for superoxide dismutase reveal a copper cluster at a domain III dimer interface.

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.

SpaC and NisC, the cyclases involved in subtilin and nisin biosynthesis, are zinc proteins.

Lantibiotics are peptide-derived antimicrobial agents that are ribosomally synthesized and posttranslationally modified by a multienzyme complex to their biologically active forms. Nisin has attracted much attention recently due to its novel mechanism of action including specific binding to the bacterial cell wall precursor lipid II, followed by membrane permeabilization. Nisin has been commercially used as a food preservative, while other lantibiotics show promising activity against bacterial infections. The posttranslational modifications are believed to be carried out by a multienzyme complex. At present the enzymes catalyzing the formation of the lantibiotic signature structural motifs, dehydroalanine (Dha), dehydrobutyrine (Dhb), lanthionine (Ln), and methyllanthionine (MeLn), are poorly characterized. In an effort to gain insight into the mechanism by which lantibiotics are biosynthesized, the cyclase enzymes involved in the synthesis of nisin and subtilin (NisC and SpaC, respectively) have been cloned, expressed, and purified. Both proteins exist as monomers in solution and contain a stoichiometric zinc atom. EXAFS data on SpaC and a C349A mutant are in line with two cysteine ligands to the metal in the wild-type enzyme with possibly two additional histidines. The two cysteine ligands are likely Cys303 and Cys349 on the basis of sequence alignments and EXAFS data. The metal may function to activate the cysteine thiol of the peptide substrate toward intramolecular Michael addition to the dehydroalanine and dehydrobutyrine residues in the peptide.