Protein Refinement and Engineering

                 
   

 

Our research is focussed on the exciting interface between chemistry and biology. This area of research is crucial for understanding both the molecular basis of biological phenomena and for the utilisation of biological materials. Projects usually start with structure determination using diffraction techniques. Following this, structures are used to rationalise protein behaviour and to design experiments that further probe the intimate relationship between structure and function. Our current work has focussed on four systems:
1) Signal transduction: The GlnK and PII proteins
2) Light stimulated regulation: Chloroplast malate dehydrogenase

3) Cell receptors: the b subunit of the Il5 receptor

   

Principal Investigator

David Ollis

Research School of Chemistry

Co-Investigators

Paul Carr

Denis Verger

Cy Jeffries

Research School of Chemistry

Projects

s06 - VPP, PC

     
               
                 

     
                 

What are the results to date and the future of the work?

The Structure of the GlnK Protein

GlnK and the PII proteins are signal transduction proteins involved in the regulation of glutamine synthetase (GS), a key element in the regulation of nitrogen uptake by Escherichia coli. The properties of GlnK and PII are very similar. PII is used by the cell as an indicator of nitrogen sufficiency and, as such, is modified by the bi-functional nitrogen sensor protein, urdiylyl transferase/removase (UT/UR). Apart from interacting with other proteins, PII also binds two affectors; ATP and a-ketoglutarate. Its affinity for each effector is in the micro-molar range, but varies with the presence of the other molecule. Under normal conditions, both ATP and a-ketoglutarate are present at millimolar levels so that PII should exist as a ternary complex.

We obtained the high resolution structure of PII as a complex with ATP. The ATP was found to occupy a cleft on the side of the molecule while the recognition loop of the protein was disordered. This suggests that the loop may be flexible in solution and that this flexibility may be important for function. When the PII sequences from bacteria were aligned, the residues forming the ATP binding site were found to be highly conserved. This suggests that the ATP binding site has an important function.

                 
- Appendix A

 
 

       

Regulation of plant malate dehydrogenase.

The NADP-malate dehydrogenases from chloroplasts undergo rapid and reversible light-dependent activation. These enzymes form part of the C4 pathway of photosynthesis in monocot plants such as maize and the dicot plants such as Flaveria bidentis. Light activation comes about by the photosynthetic dependent reduction of disulphide bonds. The purified oxidised enzyme from F. bidentis is inactive but can be activated many thousand-fold by thiol reducing agents. This regulatory mechanism is similar to the covalent regulation by protein phosphorylation that occurs in other organisms.

Chloroplast NADP-malate dehydrogenases have a similar sequence to the NAD-malate dehydrogenases that occur in the cytoplasm of bacteria, plants and animals. However, the plant enzymes also contain N- and C-terminal extensions that are not found in these other enzymes. The extensions contain cysteines that form the regulatory disulphides. The molecular basis for the dramatic change in activity between oxidised and reduced NADP-malate dehydrogenase is not well understood.

Crystals were obtained of a chloroplast NADP-dependent malate dehydrogenase from F. bidentis. The diffraction data to 2.8Å have been collected and the structure has been solved by a combination of molecular replacement and isomorphous replacement methods. The structure shows the central catalytic domain with the peptide extensions responsible for regulation. The extensions contain cysteines that form the regulatory disulphides. The C-terminal extension was found to occupy the active site so that catalysis would not be possible.

The structure of a cell surface receptor

In vivo studies have established that the cytokine, interleukin-5 (IL-5), is a regulator of growth, differentiation and activation of the white blood cell eosinophils. These cells are of major importance in the body's response to invasion by parasites and asthma inducing areoallegens. They respond to IL-5 through a membrane bound receptor that consists of an IL-5 specific a chain and a b chain that is shared with the cytokines IL-3 and GM-CSF. The structure of IL-5 is know and a model of its comlex with the a chain of the receptor can be modelled using other cytokine / receptor complexs. The structure of the b-chain is not know, but would be extremely useful in the design of anti-asthma drugs.

In collaboration with the Ian Young of the JCSMR, the extracellular portion of the b chain of interleukin-5 (IL-5) receptor has been crystallised and X-ray diffraction data have been collected to a resolution of 3 Å. Difficulties in the preparation of heavy atom derivatives have been overcome by the production of cystein enriched mutant forms of the protein. Electron density maps show the location of domains and construction of an atomic model is in progress.

What computational techniques are used?

The supercomputer is now used in all aspects of structure determination: data processing, structure determination and structure refinement. Data processing involves the analysis of diffraction images while structure determination involves a variety of techniques. This work is currently done with the CCP4 programs. The most computer intensive part of our work is structure refinement for which the X-plor program is used.

       
Appendix A -

       

     

Publications

K. Macpherson, A.R. Ashton, P.D. Carr, S.J. Trevanion, D. Verger & D.L. Ollis, Crystallisation and preliminary crystallographic analysis of chloroplast NADP- dependent malate dehydrogenase from Flaveria bidentis. Acta Cryst. D (1998) D54, 654-656.

Y. Xu, P.D. Carr, J.M. Guss, and D.L. Ollis, The crystal structure of Bikunin from the Inter-a- Inhibitor Complex: a Serine Proteinase Inhibitor with two Kuntz Domains., J. Mol. Biol. (1998) 276 (5) 955-966.

K.H.R. Macpherson, Y. Xu, E. Cheah, P.D. Carr, W. van Heeswijk, H. Westerhoff, E. Laque, S.G. Vasudevan and D.L. Ollis, Crystallization and preliminary X-ray analysis of Escherichia coli GlnK, Acta Cryst. D. (1998) D54996-998.

Y. Xu, E. Cheah, P.D. Carr, W. van Heeswijk, H. Westerhoff, E. Laque, S.G. *Vasudevan and D.L. Ollis, GlnK, a PII - homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition. J.Mol Biol (1998) 282 (1) 149-165.

     
- Appendix A