Researchers:
Dr Chloe Singleton
John Holmes

 

Funding:
BBSRC

 

 

Copper trafficking proteins of Bacillus subtilis

 

 

Collaborators:

Dr Andrew Hemmings (UEA) and Dr Jen Cavet (University of Manchester)

Researchers:

Chloe Singleton
Liang Zhou

Funding:

BBSRC

Copper plays an essential role in many cellular processes (eg respiration and photosynthesis). One example of copper enzymes in action that we are all familiar with it fruit browning. When you expose the flesh of a fruit to air, it quickly becomes brown. This is due to a copper enzyme called tyrosinase which oxidises tyrosine to eventually form pigments.  As with iron, copper is also potentially extremely toxic. This is due to its ability to redox cycle and catalyse the formation of hydroxyl radicals via Haber-Weiss like chemistry, and its ability to displace native metals from protein sites.

As well as there being conditions such as Menkes' and Wilson's diseases that result from a breakdown in copper transport, it is becoming clear that copper is an important factor in the development of a wide range of neurological disorders in humans, including Alzheimer’s and Parkinson’s diseases.  Amyloid precursor proteins from a variety of species have been shown to bind copper and this may promote aggregation leading to plaque formation.  The human prion protein is a copper-binding protein in its normal conformation, suggesting that it may have a role in brain copper metabolism.  In diseases such as Alzheimer’s, Parkinson’s and CJD it appears, therefore, that copper trafficking has gone wrong, and to understand such processes it is essential to understand how copper is  handled in normally functioning cells.

We are studying two proteins, CopZ and CopA, that are involved in trafficking copper around and out of the Gram-positive model bacterium Bacillus subtilis.  We are using a combination of spectroscopic, bioanalytical and structural methods to understand how these proteins bind copper and interact with one another to effect copper export. 

• Radford, D. S., Kihlken, M. A., Borrelly, G. P. M., Harwood, C. R., Le Brun, N. E. and Cavet, J. S. (2003) FEMS Microbiol. Lett. 220, 105-112.
• Kihlken, M. A., Leech, A. P. and Le Brun, N. E. (2002) Biochem. J. 368, 729-739.

The oxygen-sensing global transcription regulator, FNR

Collaborators:

Prof Andrew Thomson and Dr Myles Cheesman (UEA) and Dr Jeff Green (University of Sheffield) 

 

Researchers:

Dr Jason Crack

Funding:

BBSRC

In terms of survival, bacteria are extremely adaptable.  For example, many, including the model Gram-negative bacterium Escherichia coli, can grow in the presence and absence of oxygen.  The cellular machineries that enable it to do this are distinct under the two sets of conditions and so the cell must have a mechanism of sensing the oxygen concentration such that, when it drops, genes encoding anaerobic respiratory enzymes can be switched on (and vice versa).  In E. coli and many other bacteria, oxygen is sensed through the transcription regulatory protein FNR.  In the absence of oxygen the protein is dimeric, contains a [4Fe-4S] cluster in each monomer, and adopts a conformation that enables it to bind to specific operator sequences of DNA, and thus regulates the transcription of many genes.  Exposure to oxygen causes the conversion of the [4Fe-4S] cluster into a [2Fe-2S] cluster, inducing a conformational change that results in dissociation of the protein into monomers and unable to bind specifically to DNA.

We are using kinetic and spectroscopic methods to understand the mechanism by which the reaction with oxygen proceeds. 

• Crack, J. C., Green, J., Le Brun, N. E. and Thomson, A. J. (2006)  J. Biol. Chem., 281, 18909-18913

The role of ResA, an extra-cytoplasmic disulfide reductase, in cytochrome c assembly

 

 

Collaborators:

Prof Lars Hederstedt (Lund University, Sweden)

Researchers:

Dr Allison Lewin
Dr Allister Crow
Chris Hodson

Funding:

BBSRC 

C-type cytochromes play important roles in many cellular processes, such as electron transfer in respiration and photosynthesis, and signalling in programmed cell death (apoptosis). As cytochromes, they contain one or more heme C groups. Heme C is different to other types of heme because it is covalently attached to the protein via, usually, two thioether bonds between cysteine residues of the protein and the vinyl side chains of the heme ring (see above left). The cysteine residues are found in the motif Cys-Xxx-Xxx-Cys-His, where Xxx indicates any amino acid residue.   We are studying the mechanisms by which c-type cytochromes are assembled in the Gram-positive bacterium  Bacillus subtilis. Attachment of the heme occurs on the outside of the cytoplasmic membrane and requires that the cysteines of the  heme C-binding motif are in the reduced state.  Using genetic and biochemical methods, we have shown that the membrane-anchored protein ResA is required for the reduction of the cysteine side chains from the disulfide (oxidised) to the dithiol (reduced) state.  Using genetic, spectroscopic/biochemical and structural methods we are now investigating the mechanism by which ResA achieves this specific reduction reaction.

• Crow, A., Le Brun, N. E. and Oubrie, A. (2005) Biochem. Soc. Trans., 33, 149-151.
• Crow, A., Acheson, R. M., Le Brun, N. E. and Oubrie, A. (2004) J. Biol. Chem., 279, 23654-23660.
• Erlendsson, L. S., Acheson, R. M., Hederstedt, L. and Le Brun, N. E. (2003) J. Biol. Chem. 278, 17852-17858.

Extra-cytoplasmic thiol-disulfide oxidoreductases (TDORs) of Bacillus subtilis

 

 

Collaborators:

Prof Lars Hederstedt (Lund University, Sweden)

Researchers:

Dr Allister Crow
Dr Allison Lewin
Chris Hodson

Funding:

Wellcome Trust

Disulfide bonds formed between pairs of cysteine residues are crucial for stability in many proteins, while in others, such bonds can prevent folding into the correct three-dimensional structure. In order to regulate formation of required disulfide bonds, as well as breaking of dysfunctional ones, intricate disulfide bond regulatory systems have evolved. These involve enzymes of the thiol-disulfide oxidoreductases (TDOR) family, which contain cysteine residues often arranged in a thioredoxin-motif (Cys-Xxx-Xxx-Cys). These enzymes interact with two or more substrate proteins and redox cycle between disulfide bonded (oxidised) and dithiol (reduced) states with the exchange of two electrons and two protons. This proceeds via a mixed disulfide intermediate with a disulfide bond between one cysteinyl in the substrate protein and one in the oxidoreductase, see the above Figure.  Thiol-disulfide oxidoreductases (TDORs) are common in both Gram-negative and Gram-positive bacteria.   Important roles of TDORs in the expression of virulence factors such as surface components and exotoxins have so far only been demonstrated for Gram-negative bacteria. However, many Gram-positive bacteria also secrete exotoxins and other proteins of importance for e.g. virulence.  Examples of Gram-positive virulence factors that contain disulfide bonds include the botulism and tetanus toxins. It is estimated that ~60% of the ~160 predicted secreted proteins of M. tuberculosis contains at least one cysteine residue. Many of these proteins will interact with a TDOR at least once during protein folding.

Using the Gram-positive bacterium Bacillus subtilis as a model, we are applying genetic, biochemical/spectroscopic and structural methods aimed towards a detailed understanding of the physiological functions and mechanisms of action of the TDORs on the extra-cytoplasmic side of the membrane in aerobic, Gram-positive bacteria.  In addition to the reductive TDOR ResA (which is the subject of a separate project, see above), this involves studies of the oxidative TDOR BdbD (involved in the formation of disulfide bonds), and another reductive TDOR, StoA, which is required for sporulation.  Understanding the roles of these proteins will significantly advance our understanding of extra-cytoplasmic thiol-disulfide redox processes.