Structural Biology

Major Research Interests

The mission of the structural biology research team is to unravel structural and functional relationships of plant proteins participating during normal plant development and during plant development under environmental stresses.

Specifically, we are interested in alpha-helical membrane proteins that mediate transport of ions across plant membranes, as well as in ‘modus operandi’ of enzymes, transcription factors and other proteins. Examples include a boron transporter, Na+/K+ ion HKT transporters, bZip and HDZip transcription factors, and enzymes involved in grain development.

Additionally, our goals are to contribute to cell-based and cell-free protein production platforms (Hrmova and Fincher, 2009a; Luang et al., 2010), and we also develop nanotechnology approaches to membrane protein structure. The latter approaches would allow us to prepare membrane proteins in crystalline states, so we could ultimately solve their three-dimensional (3D) structures.

Finally, we perform protein modeling and in-silico mining studies to pin-point structural and functional relationships, and how to engineer variant proteins with modified biological functions (Hrmova and Fincher, 2009a). The acquired knowledge could be important for improvement of cereals’ tolerance to environmental stresses.

Key Research Projects

Barley borate transporter (HvBot1) involved in boron toxicity

Boric acid toxicity is widespread in semi-arid regions of the world and is difficult to manage agronomically. In barley, boric acid tolerance is most commonly associated with a limited net entry of boric acid into roots and an ability of leaves to dispose excess of it. The boric acid tolerance gene HvBot1 was recently identified in a boric acid tolerant barley landrace Sahara (Sutton et al., 2007). This tolerant variety contains four times as many HvBot1 gene copies as intolerant varieties and produces elevated levels of HvBot1 gene transcript. The cDNA of HvBot1 translates into a 666-residue protein and its 3D model indicates that HvBot1 spans the membrane 10-12 times.

The boron tolerance is also mediated by the decreased expression of the multifunctional aquaporin HvNIP2;1 (Schnurbusch et al., 2010), so HvNIP2;1 along with HvBot1 are important determinants of boron toxicity tolerance in barley.

Molecular model of HvBot1

Wheat and rice HKT Na+/K+ transporters involved in salinity

Salinity tolerance in plants is inversely related to the extent of Na+ accumulation in shoots in the major cereals such as wheat and rice. In Arabidopsis and rice data pin-point at a central role for the HKT gene family of Na+ and Na+/K+ transporters in controlling Na+ accumulation, and hence salinity tolerance. The focus of this project is on OsHKT1;5 and TaHKT1;5 that represent low- and high-affinity Na+ transporters, respectively. The molecular basis of the Na+/K+ transport in the plant HKT-type transporters is essentially unknown, and the goal is to envisage the basis of Na+/K+ ion selectivity and how these transporters regulate Na+/K+ homeostasis. The molecular models of OsHKT1;5 from rice Nippobore and Pokkali varieties along with transcriptomics and physiological data suggest possible Na+ exclusion mechanisms in rice.

Molecular model of OsHKT1;5: an entry into the pore.

Wheat transcription factors, lipid-binding proteins and defensins involved in drought and grain development

Drought often occurs during flowering and early grain development, leading to disruption of normal fertilization and inducing abortion of early grain, with substantial losses in grain yield. One of the first events of stress response is the transcriptional regulation of drought related genes (Harris et al., 2011).

Studies have shown that members of several transcription factors are up- or down-regulated in plant tissue during drought, for example bZip, HDZip, ERF and DREB (Harris et al., 2011). Through molecular modeling we investigate functional roles of the individual structural components in transcription factors TabZip, TaHDZip and TaERF, lipid-binding proteins TaPR60 and TaPR61 (Kovalchuk et al., 2009; 2012), and a defensin TaPRPI (Kovalchuk et al., 2010), and how sequence diversities reflect proteins’ functional roles and their biological activities.

Molecular model of TaDREB3 in complex with DNA.

Crystal structures of barley exoglucanase HvExoI involved in grain germination

The emphasis is on a soluble β-d-glucan exohydrolase (HvExoI) enzyme from barley that plays key roles in grain development. Here we aim to define in atomic details enzyme’s catalytic mechanism, and thermodynamic and structural determinants of substrate recognition. We have solved around twenty 3D structures of HvExoI in complex with mechanism-based inhibitors, substrate analogues and transition state mimics, and used molecular dynamics to elucidate various aspects of enzyme catalysis and specificity (Varghese et al., 1999; Hrmova et al., 2001; 2002; 2004; 2005).

Currently, we investigate variant forms of HvExoI (Luang et al., 2010, 2011) and their binary complexes with thio-analogues that were acquired at the Australian Synchrotron. Crystal structures of variant forms of HvExoI will address mechanisms of catalysis and structural basis of broad substrate specificity and will lead to designer enzymes (Hrmova and Fincher, 2007).

Crystal structure of HvExoI: cartoon (left) and surface (right) projections.

Development of heterologous protein production technologies

The focus of the group is also in development of heterologous protein production technologies through cell-based and cell-free platforms. We have constructed cDNA fusions of large alpha-helical membrane proteins (curdlan synthase CrdS, HvBot1, TaHKT1;5) and produced these proteins in high yields in a wheat germ cell-free system (WG-CFS) with a range of surfactants, surfactant peptides and lipids. These proteins are being tested for functionality such as substrate selectivity and kinetic properties. We have also assembled nanodiscs mimicking native membranes with the HvBot1, TaHKT1;5 and CrdS proteins. CrdS and HvBot1 liposomes and nanodiscs are analysed at the Australian Synchrotron to de-convolute proteins’ molecular characteristics. 

For more information please contact

The group leader of the structural biology research group Associate Professor Maria Hrmova
maria.hrmova@acpfg.com.au

Research personnel

Associate Professor Maria Hrmova (Group leader)
Dr Jeab Luang (Postdoctoral Fellow)
Dr Sona Garajova (Group of Eight Fellow)
Nadim Shadiac (Research Associate)
Amritha Amalraj (Research Associate)
Ramya Sampath (Research Associate)
Yagnesh Nagarajan (PhD student)
Shane Waters (PhD student)

Collaborators

Dr Sergiy Lopato and Professor Peter Langridge, ACPFG - Drought Genetics Group (Australia)
Dr Victor Streltsov and Dr Jose Varghese, CSIRO - Material Science and Engineering (Australia)
Dr Igor Tvaroska, Institute of Chemistry, Slovak Academy of Sciences (Slovak Republic)
Professor Werner Kuehlbrandt, Max Planck Institute of Biophysics (Germany)
Professor Nico Voelcker, University of South Australia (Australia)
Dr Ingo Koeper, and Flinders University (Australia)
Dr Haydyn Martens, Australian Synchrotron, SAXS/WAXS (Australia))

Key publications

  • Harris J, Hrmova M, Lopato S, Langridge P (2011) Phytochemistry. New Phytol 190, 823-837. Tansley review.
  • Luang S, Ketudat Cairns JR, Streltsov VA, Hrmova M (2010) International Journal of Molecular Sciences 11, 2759-2769.
  • Luang S, Hrmova M, Ketudat Cairns JR (2010) Prot Express Purif 73, 90-98.
  • Hrmova M, Fincher GB (2009a) Functional genomics and structural biology in the definition of gene function. Plant Genomics, Humana Press Inc., USA. Meth Mol Biol 513, 199-227.
  • Sutton T, Baumann U, Hayes J, Shi B-J, Collins NC, Schnurbusch T, Hay A, Mayo G, Pallotta M, Tester M, Langridge P (2007) Science 318, 1446-1449.
  • Schnurbusch T, Hayes J, Hrmova M, Baumann U, Ramesh SA, Tyerman SD, Langridge P, Sutton T (2010) Plant Physiology 153, 1706-1715.
  • Kovalchuk N, Smith J, Pallotta M, Singh R, Hrmova M, Langridge P, Lopato S (2009) Plant Mol Biol 71, 81-98.
  • Kovalchuk N, Li M, Wittek F, Reid N, Shirley N, Ismagul A, Eliby S, Johnson A, Milligan AS, Hrmova M, Langridge P, Lopato S (2010) J Biotech 8, 47-64.
  • Kovalchuk N, Smith J, Bazanova N, Pyvovarenko T, Singh R, Shirley N, Ismagul A, Johnson A, Milligan AS, Hrmova M, Langridge P, Lopato S (2012) J Exp Bot 63, 2025-2040.
  • Varghese JN, Hrmova M, Fincher GB (1999) Structure 7, 179-190.
  • Hrmova M, Varghese JN, DeGori R, Smith BJ, Driguez H, Fincher GB (2001) Structure 9, 1005-1016.
  • Hrmova M, De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, Fincher GB (2002) Plant Cell 14, 1033-1052.
  • Hrmova M, De Gori R, Smith BJ, Vasella A, Varghese JN, Fincher GB (2004) JBC 279, 4970-4980.
  • Hrmova M, Streltsov VA, Smith BJ, Vasella A, Varghese JN, Fincher GB (2005) Biochemistry (USA) 44, 16529-16539.
  • Hrmova M, Fincher GB (2007) Carbohydr Res 342, 1613-1623.
  • Hrmova M, Farkas V, Lahnstein J, Fincher GB (2007) JBC 282, 12951-12962.
  • Hrmova M, Harvey AJ, Lahnstein J, Wischmann B, Kaewthai N, Ezcurra I, Teeri TT, Fincher GB (2009) FEBS J 276, 437-456.

A Laue diffraction image of HvExoI taken at the Advanced Photon Source (Chicago, USA).

 

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