Structural basis of molecular function of plant proteins

Major Research Interests

The mission of our 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 solutes and ions across plant membranes, as well as in 'modus operandi' of enzymes, transcription factors and other proteins. Examples include boron and silicon transporters, Na+/K+ ion HKT transporters, ERF and DREB transcription factors, and cell wall enzymes (Amalraj et al., 2015; Yadav et al., 2015; Zhang et al., 2015; Borisjuk et al., 2014; Hrmova and Lopato, 2014; Lopato et al., 2014). Additionally, our goals are in contributing to recombinant protein expression and purification (Hrmova and Fincher, 2009a; Luang et al., 2010; Hrmova et al., 2010).

We are also interested in nanotechnology of membrane proteins and intend to incorporate them in liposomes and nanodiscs to generate functional lipo-protein particles. Further, our goals are to prepare proteins in crystalline states, so we could ultimately solve their three-dimensional (3D) structures. Finally, in conjunction with our experimental approaches, we perform extensive 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 precise structural information would allow us to perform rational design to improve cereal tolerance to environmental stresses.

Key Research Projects

(1) Solute transporters HvBot1 and HvNIP2;1 from barley

Molecular model of HvNIP2;1
with BA and water molecules.

Boric acid (BA) toxicity is widespread in semi-arid regions of the world and is difficult to manage agronomically. In barley, BA tolerance is most commonly associated with a limited net entry of BA into roots and an ability of leaves to dispose of excess BA. The BA tolerance gene Bot1 was recently identified in a BA tolerant barley landrace Sahara (Sutton et al., 2007). The cDNA of Bot1 translates into an integral membrane protein of 666 amino acid residues.

Silicic acid (SA) is a major mineral component of cereals and it helps plants withstand abiotic stresses and pathogen attacks. One of proteins that mediates transport of SA in cereals is the Lsi1 transporter that was classified as an aquaporin. A similarly functioning HvNIP2;1 protein has recently been identified in barley, and its 3D molecular model indicates that it could transport SA, BA, germanic acid and water (Schnurbusch et al., 2010). Plant multi-functional aquaporins fall into a clade distinct to the structurally characterized aquaporins and therefore their 3D structures are desired.

(2) Na+/K+ ion transporters OsHKT1;5 from wheat and rice

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

Salinity tolerance in plants is inversely related to the extent of Na+ accumulation in shoots, notably in the major cereals such as wheat and rice. In Arabidopsis and rice there is evidence indicating 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 and Na+/K+ symporters. 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 in these integral membrane proteins 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.

(3) Transcription factors, lipid-binding proteins and defensins from wheat

Molecular model of TaDREB3
in complex with DNA.

We are interested in molecular mechanisms, how wheat proteins play roles in drought-responsive gene regulation, bind lipids and function as defensins. Through molecular modeling we investigate functional roles of individual structural components in transcription factors (TaERF and TaDREB), lipid-binding proteins (TaPR60) and defensins (TaPRPI), and how sequence diversities reflect proteins' functional roles and biological activities.

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. Studies have shown that members of several families of transcription factors are up- or down-regulated during drought, for example AP2/ERF and DREB transcription factors.

The TaPR60 gene from wheat encodes a small cysteine-rich protein. Examination of a 3D model of TaPR60 suggested that this protein could be involved in binding of lipid-like molecules (Kovalchuk et al., 2009).

The cDNAs of seven defensins have been cloned from wheat and rice (Kovalchuk et al., 2010). All wheat and rice promoters of defensins are strongly induced by wounding in the leaf tissue of transgenic rice plants. These promoters could be useful for specific targeting and accumulation of pathogen resistance genes to young vulnerable tissues of a developing and germinating grain, and for a swift activation in wounded areas.

(4) Cell-wall biosynthetic, hydrolytic and re-modeling enzymes from barley

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

The emphasis is on soluble and integral membrane enzymes that play key roles in biosynthesis, degradation and remodelling of plant cell-walls (Hrmova and Fincher, 2009b).

From hydrolytic enzymes, the foci of interest are β-Dglucan exohydrolases (HvExo) from barley. Here we aim to define in atomic details their 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).

Realization of these objectives will provide fundamental information on the catalytic mechanisms of polysaccharide synthesis (Burton et al., 2006; Hrmova and Fincher, 2009b) degradation (Hrmova and Fincher, 2007) and re-modeling (Hrmova et al., 2007; 2009; Vaaje-Kolstad et al., 2010).

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Research personnel

Professor Maria Hrmova (Group leader)
Dr Jeab Luang (Postdoctoral Fellow)
Dr Syed Sarfraz (Postdoctoral Fellow)
Huihui Bi (PhD student)
Shane Waters (PhD student)


Professor Steve Tyerman, ARC Centre of Excellence in Plant Energy Biology, University of Adelaide, Australia
Professor Nicolas Voelcker, Mawson Institute, University of South Australia, Australia
Professor Yaroslava Yingling and Dr Abhishek Singh, North Carolina State University, USA
Dr Victor Streltsov, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Australia
Dr Haydyn Mertens, BIOSAXS Group, EMBL Hamburg, Germany
Dr Ingo Köper, Flinders Centre for Nanoscale Science and Technology, Flinders University, Australia
Dr Tim Sutton, South Australian Research and Development Institute, Australia

Key selected works

Nagarajan Y, Rongala J, Luang S, Shadiac N, Hayes J, Sutton T, Gilliham M, Tyerman SD, McPhee G, Voelcker NH, Mertens HDT, Kirby NM, Sing A, Lee J-G, Yingling YG, Hrmova M (2016) A barley efflux transporter operates in a Na+-dependent manner, as revealed through a multidisciplinary platform. Plant Cell 28, 202-218.

Kovalchuk N, Chew W, Sornaraj P, Borisjuk N, Yang N, Singh R, Bazanova N, Shavrukov Y, Guendel A, Munz E, Borisjuk L, Langridge P, Hrmova M, Lopato S (2016) The homeodomain transcription factor TaHDZipI-2 from wheat regulates frost tolerance, flowering time and spike development in transgenic barley. New Phytologist, accepted on 03 February 2016.

Harris JC, Sornaraj P, Taylor M, Bazanova N, Baumann U, Lovell B, Langridge P, Lopato S, Hrmova M (2016) Molecular interaction of the γ-Clade Homeodomain-Leucine Zipper class I transcription factors during the wheat response to water deficit. Plant Molecular Biology, doi 10.1007/s11103-015-0427-6.

Li B, Evrard A, Qiu J, Johnson AAT, Baumann U, Birnbaum KD, Hrmova M, Mayo GM, Jha D, Henderson S, Tester M, Gilliham M, Roy SJ (2016) Identification of a stelar-localised transport protein that facilitates root-to-shoot transfer of chloride in Arabidopsis. Plant Physiology, doi:10.1104/pp.15.01163.

Sornaraj P, Luang S, Lopato S, Hrmova M (2016) Basic leucine zipper (bZIP) transcription factors involved in abiotic stresses: A molecular model of a wheat bZIP factor and implications of its structure in function. Biochimica et Biophysica Acta - General Subjects 1850, 46-56.

Yadav D, Shavrukov Y, Bazanova N, Chirkova L, Borisjuk N, Kovalchuk N, Ismagul A, Parent B, Hrmova M, Langridge P, Lopato S (2015) Constitutive over-expression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield. Journal of Experimental Botany, doi: 10.1093/jxb/erv370.

Amalraj A, Luang S, Kumar M, Sornaraj P, Eini O, Kovalchuk N, Bazanova N, Li Y, Yang N, Eliby S, Langridge P, Hrmova M, Lopato S (2015) Change of function of the wheat stress-responsive transcriptional repressor TaRAP2.1L by repressor motif modification. Plant Biotechnology Journal 14, 820-832, doi: 10.1111/pbi.12432.

Zhang H, Luo M, Day R, Talbot MJ, Ivanova A, Ashton AR, Chaudhury AM, Macknight R, Hrmova M, Koltunow AM (2015) Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation and seed development in Arabidopsis. Journal of Experimental Botany, doi: 10.1093/jxb/erv296.

Tankrathok A, Iglesias-Fernández J, Williams RJ, Pengthaisong S, Baiya S, Hakki Z, Robinson RC, Hrmova M, Rovira C, Williams SJ, Ketudat Cairns JR (2015) A single glycosidase harnesses different transition state conformations for hydrolysis of mannosides and glucosides. American Chemical Society: Catalysis 5, 6041-6051.

Li M, Lopato S, Hrmova M, Pickering M, Shirley N, Koltunow AM, Langridge P (2014) Expression patterns and protein structure of a lipid transfer protein END1 from Arabidopsis. Planta 240, 1319-1334.

Lopato S, Borisjuk N, Langridge P, Hrmova M (2014) Endosperm transfer cell-specific genes and proteins: structure, function and applications in biotechnology. Frontiers in Plant Science 5, article 64, 1-14.

Borisjuk N, Hrmova M, Lopato S (2014) Transcriptional regulation of cuticle biosynthesis. Biotechnology Advances 32, 526-540.

Hrmova M, Lopato S (2014) Enhancing abiotic stress tolerance in plants by modulating properties of stress responsive transcription factors. In: Genomics of Plant Genetic Resources (Tuberosa R, Graner A, Frison E, eds.), Springer Netherlands, 515 pp, Volume 2, Part II: Crop productivity, food security and nutritional quality, pp 291-316. Invited review.

Tankrathok A, Luang S, Robinson R, Kimura A, Rovira C, Hrmova M, Ketudat Cairns J (2013) Structural analysis and insights into glycon specificity of the rice GH1 Os7BGlu26 β-d-mannosidase. Acta Crystallographica Section D69, 2124-2135.

Chew W, Hrmova M, Lopato S (2013) Role of HD-Zip IV transcription factors in plant development and plant protection from deleterious environmental factors. International Journal of Molecular Sciences 14, 8122-8147.

Waters S, Gilliham M, Hrmova M (2013) Plant high affinity potassium (HKT) transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity. International Journal of Molecular Sciences 14, 7660-7680.

Eini O, Yang N, Pyvovarenko T, Pillman K, Bazanova N, Tikhomirov N, Eliby S, Shirley N, Sivasankar S, Tingey S, Langridge P, Hrmova M, Lopato S (2013) Complex regulation by Apetala2 domain-containing transcription factors revealed through analysis of the stress-responsive TdCor410b promoter from durum wheat. PLoS ONE 8, e58713.

Shadiac N, Nagarajan Y, Waters S, Hrmova M (2013) The close allies in membrane protein research: cell-free synthesis and nanotechnology. Molecular Membrane Biology 30, 229-245.

Periasamy A, Shadiac N, Amalraj A, Garajová S, Nagarajan Y, Waters S, Mertens HD, Hrmova M (2013) Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs. Biochimica et Biophysica Acta - Biomembranes 1828, 743-757.

Cotsaftis O, Plett D, Shirley N, Tester M, Hrmova M (2012) A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing. PLoS ONE 7, e39865.

Fernandez i Marti A, Wirthensohn M, Alonso J, Socias i Company R, Hrmova M (2012) Molecular modelling of S-RNases involved in almond self-incompatibility. Frontiers in Crop Science and Horticulture 3, 1-4.

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) TdPR61 is a marker for nutrient fluxes and transport pathways in the endosperm and embryo of wheat, barley and rice. Journal of Experimental Botany 63, 2025-2040. Feature article with a front cover photograph.

Harris J, Hrmova M, Lopato S, Langridge P (2011) Modulation of plant growth by HDZip class I and II transcription factors in response to environmental stimuli. New Phytologist 190, 823-837. Tansley review.

Rivandi J, Miyazaki J, Hrmova M, Pallotta M, Tester M, Collins NC (2011) A SOS3 homologue maps to HvNax4, a barley locus controlling an environment-dependent Na+ exclusion trait. Journal of Experimental Botany 62, 1201-1211. Feature article with a front cover photograph.

Stone BA, Jacobs AK, Hrmova M, Burton RA, Fincher GB (2011) The biosynthesis of plant cell wall and related polysaccharides by enzymes of the GT2 and GT48 families. In: Plant Polysaccharides, Series: Annual Plant Reviews 41, 109-166. Blackwell Publishing Inc, Danvers, MA, USA (Ulvskov P, ed). Invited review.

Drew DP, Hrmova M, Lunde C, Jacobs A, Tester M, Fincher GB (2011) Structural and functional analyses of PpENA1 provide insights into cation binding by type IID P-type ATPases in lower plants and fungi. Biochimica et Biophysica Acta - Biomembranes 1808, 1483-1492.

Kaiser BN, Hrmova M (2010) A glimpse at regulation of nitrogen homeostasis. Structure 18, 1395-1397. Invited commentary.

Kuntothom T, Raab M, Tvaroška I, Fort S, Pengthaisong S, Cañada FJ, Calle L, Jiménez-Barbero J, Ketudat Cairns JR, Hrmova M (2010) Binding of β-d-glucosides and β-d-mannosides by rice and barley β-d-glycosidases with distinct substrate specificities. Biochemistry (USA) 49, 8779-8793.

Luang S, Ketudat Cairns JR, Streltsov VA, Hrmova M (2010) Crystallisation of wild-type and variant forms of a recombinant β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) by macroseeding with wild-type native microcrystals and preliminary X-ray analysis. International Journal of Molecular Sciences 11, 2759-2769.

Schnurbusch T, Hayes J, Hrmova M, Baumann U, Ramesh SA, Tyerman SD, Langridge P, Sutton T (2010) Boron toxicity tolerance in barley through reduced expression of the multifunctional aquaporin, HvNIP2;1. Plant Physiology 153, 1706-1715.

Kaewthai N, Harvey AJ, Hrmova M, Brumer H, Ezcurra I, Teeri TT, Fincher GB (2010) Recombinant expression of a diversity of barley XTH genes in the yeast Pichia pastoris. Plant Biotechnology 27, 251-258.

Luang S, Hrmova M, Ketudat Cairns JR (2010) High-level expression of barley β-d-glucan exohydrolase HvExoI from a codon-optimized cDNA in Pichia pastoris. Protein Expression and Purification 73, 90-98.

Kosik O, Auburn RP, Stratilova E, Garajova S, Hrmova M, Farkas V (2010) Polysaccharide micro-arrays for screening of transglycosylase activities in plant extracts. Glycoconjugate Journal 27, 79-87.

Hanlin RL, Hrmova M, Harbertson JF, Downey MO (2010) Condensed tannin and grape cell wall interactions and impact on tannin extractability into wine - a review. Australian Journal of Grape and Wine Research 16, 173-188. Feature article with a front cover photograph.

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) Defensin promoters as tools for engineering disease resistance in cereal grains. Plant Biotechnology Journal 8, 47-64. Feature article with a front cover photograph.

Vaaje-Kolstad G, Farkas V, Fincher GB, Hrmova M (2010) Barley xyloglucan xyloglucosyl transferases bind xyloglucan-derived oligosaccharides in their acceptor binding regions in multiple conformational states. Archives of Biochemistry and Biophysics 496, 61-68.

Vaaje-Kolstad G, Farkaš V, Hrmova M, Fincher GB (2010) Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites. Biochimica et Biophysica Acta - General Subjects 1800, 674-684.

Zhang Q, Shirley NJ, Burton RA, Lahnstein J, Hrmova M, Fincher GB (2010) The genetics, transcriptional profiles and catalytic properties of UDP-α-d-xylose 4-epimerases from barley (Hordeum vulgare L.). Plant Physiology 153, 555-568.

Hrmova M, Stone BA, Fincher GB (2010) High-yield production, refolding and molecular modelling of the catalytic module of (1,3)-β-d-glucan (curdlan) synthase from Agrobacterium sp. Glycoconjugate Journal 27, 461-476.

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


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