Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans.

In Streptomyces lividans an extracytoplasmic copper-binding Sco protein plays a role in two unlinked processes: (i) initiating a morphological development switch and (ii) facilitating the co-factoring of the CuA domain of CcO (cytochrome c oxidase). How Sco obtains copper once secreted to the extracytoplasmic environment is unknown. In the present paper we report on a protein possessing an HX6MX21HXM motif that binds a single cuprous ion with subfemtomolar affinity. High-resolution X-ray structures of this extracytoplasmic copper chaperone-like protein (ECuC) in the apo- and Cu(I)-bound states reveal that the latter possesses a surface-accessible cuprous-ion-binding site located in a dish-shaped region of ß-sheet structure. A cuprous ion is transferred under a favourable thermodynamic gradient from ECuC to Sco with no back transfer occurring. The ionization properties of the cysteine residues in the Cys86xxxCys9° copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys86 in initiating an inter-complex ligand-exchange reaction with Cu(I)-ECuC. Generation of the genetic knockouts, Δsco, Δecuc and Δsco/ecuc, and subsequent in vivo assays lend support to the existence of a branched extracytoplasmic copper-trafficking pathway in S. lividans. One branch requires both Sco and to a certain extent ECuC to cofactor the CuA domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.

The hydrogen-peroxide-induced radical behaviour in human cytochrome c-phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant.

We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met(80) ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr(46) and Tyr(48), have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.

The structure of FKBP38 in complex with the MEEVD tetratricopeptide binding-motif of Hsp90.

Tetratricopeptide (TPR) domains are known protein interaction domains. We show that the TPR domain of FKBP8 selectively binds Hsp90, and interactions upstream of the conserved MEEVD motif are critical for tight binding. In contrast FKBP8 failed to bind intact Hsp70. The PPIase domain was not essential for the interaction with Hsp90 and binding was completely encompassed by the TPR domain alone. The conformation adopted by Hsp90 peptides, containing the conserved MEEVD motif, in the crystal structure were similar to that seen for the TPR domains of CHIP, AIP and Tah1. The carboxylate clamp interactions with bound Hsp90 peptide were a critical component of the interaction and mutation of Lys 307, involved in the carboxylate clamp, completely disrupted the interaction with Hsp90. FKBP8 binding to Hsp90 did not substantially influence its ATPase activity.

Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System.

Selective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions.

Dihydropyridine Derivatives Modulate Heat Shock Responses and have a Neuroprotective Effect in a Transgenic Mouse Model of Alzheimer's Disease.

Heat shock proteins (Hsps) have chaperone activity and play a pivotal role in the homeostasis of proteins by preventing misfolding, by clearing aggregated and damaged proteins from cells, and by maintaining proteins in an active state. Alzheimer's disease (AD) is thought to be caused by amyloid-ß peptide that triggers tau hyperphosphorylation, which is neurotoxic. Although proteostasis capacity declines with age and facilitates the manifestation of neurodegenerative diseases such as AD, the upregulation of chaperones improves prognosis. Our research goal is to identify potent Hsp co-inducers that enhance protein homeostasis for the treatment of AD, especially 1,4-dihydropyridine derivatives optimized for their ability to modulate cellular stress responses. Based on favorable toxicological data and Hsp co-inducing activity, LA1011 was selected for the in vivo analysis of its neuroprotective effect in the APPxPS1 mouse model of AD. Here, we report that 6 months of LA1011 administration effectively improved the spatial learning and memory functions in wild type mice and eliminated neurodegeneration in double mutant mice. Furthermore, Hsp co-inducer therapy preserves the number of neurons, increases dendritic spine density, and reduces tau pathology and amyloid plaque formation in transgenic AD mice. In conclusion, the Hsp co-inducer LA1011 is neuroprotective and therefore is a potential pharmaceutical candidate for the therapy of neurodegenerative diseases, particularly AD.

The role of the Cys-X-X-X-Cys motif on the kinetics of cupric ion loading to the Streptomyces lividans Sco protein.

The mechanisms and spectroscopic properties generated by intermediate states upon cupric ion binding to flexible peptide motifs in proteins are of considerable interest. One such motif is the Cys-X-X-X-Cys motif characteristic to members of the Sco family of proteins. In the antibiotic producing bacterium, Streptomyces lividans, a role for its Sco protein (Sco(Sl)) as a cupric metallochaperone to the extracytoplasmic CuA domain of cytochrome c oxidase has been revealed. Stopped-flow kinetic studies have revealed a mechanism of cupric ion capture by Sco(Sl), which passes through a monothiolate intermediate, with distinct spectral features. In the present study we have used two site directed mutants of Sco(Sl), C86A and C90A, to determine which Cys in the CXXXC motif acts as the capture ligand. Comparison of kinetic and thermodynamic parameters obtained from cupric ion binding to the C86A and C90A mutants clearly indicate that Cys86 is the capture ligand and this finding can be reconciled with structural data. At subsaturating levels of cupric ions both mutants bind copper rapidly, but the absorbance properties are distinctly different from wild type Sco(Sl). This is most extreme for the C86A mutant where the Cys90 thiolate bond is considered to be weaker than the Cys86 thiolate bond in the C90A mutant. We put forward an explanation for this behaviour whereby we propose that the cupric ion is moving to a second site with no thiolate coordination.

Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein.

Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes, a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. We report on the Sco-like protein from Streptomyces lividans (Sco(Sl)) and present a series of experiments that firmly establish a role for Sco(Sl) as a copper metallochaperone as opposed to a role as a thiol-disulphide reductase that has been assigned to other bacterial Sco proteins. Under low copper concentrations, a Δsco mutant in S. lividans displays two phenotypes; the development switch between vegetative mycelium and aerial hyphae stalls and cytochrome c oxidase (CcO) activity is significantly decreased. At elevated copper levels, the development and CcO activity in the Δsco mutant are restored to wild-type levels and are thus independent of Sco(Sl). A CcO knockout reveals that morphological development is independent of CcO activity leading us to suggest that Sco(Sl) has at least two targets in S. lividans. We establish that one Sco(Sl) target is the dinuclear Cu(A) domain of CcO and it is the cupric form of Sco(Sl) that is functionally active. The mechanism of cupric ion capture by Sco(Sl) has been investigated, and an important role for a conserved His residue is identified.