Biophysical Society Thematic Meeting

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

4-POS

Board 4

Structure Determination of Enzymes Involved in Mutagenesis in Mycobacterium

Tuberculosis

Simon Broadley

, Digby Warner

2,3

, Trevor Sewell

1,3

University of Cape Town, Cape Town, Western Cape, South Africa,

University of Cape Town,

Cape Town, Western Cape, South Africa,

University of Cape Town, Cape Town, Western Cape,

South Africa.

Drug resistance in

Mycobacterium tuberculosis

(MTB) arises through the acquisition of

spontaneous mutations in antibiotic target or related genes. This places enormous importance on

the need to understand the DNA metabolic pathways in MTB, and identifies mutagenic repair

mechanisms as compelling targets for novel anti-TB drugs.

The DNA damage-inducible C family DNA polymerase, DnaE2, has been implicated in

virulence and the emergence of antibiotic-resistant MTB mutants in vivo. DnaE2 operates as part

of a three-component “mutagenic cassette” comprising ImuB - a pseudo Y-family polymerase -

and ImuA’, a RecA-like protein of unknown function.

The aim of this study is to obtain crystal structures of these proteins as well as the

dnaE1

encoded replicative polymerase in order to gain insight into the comparative geometries of the

DnaE1 and DnaE2 active sites, and to elucidate potential interacting domains in DnaE2, ImuA’,

and ImuB.

To date, ImuA’ and DnaE1 have been solubly expressed in

E. coli

with maltose-binding protein

tags. Thermostability studies (which provide an indication of proper protein folding and a

measure of stability in crystallization buffers) have been inconclusive and indicate the need for

further investigation. MTB proteins are notoriously difficult to express properly folded in large

quantities; therefore, optimised codon usage for expression of MTB genes in E.coli has been

explored to improve folding stability. Biophysics also suggests that slower expression increases

the probability of proper folding; in addition, other expression vectors, strains and conditions

have been tested. Provided purified, soluble protein is obtained in sufficient quantities,

crystallisation conditions will be screened using a mosquito pipetting robot and hits will be

optimised by exploring the crystallisation phase diagram. Crystal cryoprotectant conditions will

be optimised using the diffractometer at UCT and the final, high-quality data sets obtained at a

synchrotron.