Protein Refinement and Engineering

                   

Principal Investigator

David Ollis

Research School of Chemistry

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) The PII / GlnK proteins

2) Bikunin

3) Peptidyl-prolyl isomerase (Ppi B)

4) Chloroplast malate dehydrogenase

   

Co-Investigators

     

Paul Carr

Denis Verger

Karen Edwards

Eong Cheah

Yibin Xu

Research School of Chemistry

     
                 

     
                 

 

Projects

s06 - VPP, PC

           

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

GlnK is a signal transduction protein involved in the regulation of glutamine synthetase (GS), a key element in the regulation of nitrogen uptake by Escherichia coli. The properties of GlnK are very similar to those of the PII (GlnB) protein whose structure has been the subject of previous reports. 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). Native PII indicates the environment is nitrogen rich while PII-UMP indicates a nitrogen poor environment.

Bikunin is a protease inhibitor of about 140 amino acids that folds into two domains which are structurally similar to bovine pancreatic trypsin inhibitor (BPTI - Kunitz family). Although it is isolated from urine, the importance of Bikunin is probably related to its occurrence in blood where it part of the Inter-a-trypsin Inhibitor (ITI) complex. There are indications that it is involved in the regulation of cell growth. For example, it has been found to be more abundant in pregnant females as well as people suffering from various forms of cancer. In addition, studies with Bikunin have established that it inhibits tumour cell invasion.

   
                   
- Appendix A
 

 
                   
       

We have obtained the structure of a bacterial protein capable of catalysing the cis-trans isomerisation of peptidyl-prolyl bonds. This inter-conversion is frequently the slow step in protein folding.

The NADP-malate dehydrogenase from chloroplasts undergoes 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 enzymes from F.bidentis is inactive but can be activated many thousand-fold by thiol reducing agents. 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 enzyme 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.

In the last year we have obtained the high resolution structure of GlnK as well as the GlnK complex with ATP. The ATP was found to occupy a cleft in the side of the molecule and has an effect on the loop region of the molecule. There are two independent copies of the GlnK molecule in the crystals. Like PII, the these GlnK molecules are trimers with barrel like cores. The two GlnK molecules have similar core structures and differ significantly from PII at the C-terminus. In addition, the loops of the two GlnK molecules differ from each other and from PII. This suggests that the loops of the protein may be flexible and that this flexibility may be important for function. When the PII / GlnK sequences from bacteria are aligned, the T-loop appears to be highly conserved. Conservation of sequence usually implies structural conservation, in this case, conservation of sequence is associated with the mobility of the peptide.

The structure of bikunin has been solved using molecular replacement methods and refined to an R-factor of 20.%. The two BPTI domains are related by a rotation of about 60 degree combined with translation. Protease recognition occurs via recognition loops in the two domains. The recognition loops of the first domain are unaffected by the second domain, but those of the second domain are close to the first domain. The first domain could interfere with protease binding at the second. It has been reported that a recombinant form of the second domain inhibits human blood coagulation factor Xa and plasma kallikrein while the intact bikunin does not inhibit these two enzymes. This selective inhibition may prove to be a useful regulatory mechanism.

The structure of E. coli PPI B has been determined. Comparison of this structure with that of the peptide bound form of the protein suggests that the mobility of loops may be important in the catalytic action of the enzyme.

Crystals of chloroplast NADP-dependent malate dehydrogenase from F.bidentis have been obtained with the cofactor NADP present. The enzyme has a molecular weight of 43,000 daltons per subunit and exists as a dimer in solution. The crystals diffract to 2.8Å and belong to the space group P3221 with cell dimensions a = 148.1, c = 65.5 Å. The structure has been solved by a combination of molecular replacement and isomorphous replacement methods.

       
Appendix A -

       
     

The structure has been refined to the limits of diffraction and shows the central catalytic domain with the peptide extensions responsible for regulations.

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.

Publications

K. J. Edwards, D. L. Ollis, and N. E. Dixon, Crystal Structure of Cytoplasmic Escherichia coli Peptidyl-prolyl Isomerase: Evidence forDecreased Mobility of Loops upon Complexation, J. Mol. Biol. 271:258-265 (1997).

     
- Appendix A