Associate Professor Alok K Mitra

Academic Leader of the cryo-EM facility

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Associate Professor

Research | Current

Our research focus is to gain mechanistic insight into large protein complexes and integral membrane and membrane-associated proteins that are crucial for proper functioning of the cell. It is becoming increasingly clear that many cellular processes are carried out by multi-protein complexes or protein machines and recent genomic studies on both prokaryotic and eukaryotic organisms have indicated that over 20% of the gene products are integral membrane or membrane–associated proteins. These constitute 70% of current drug targets. Our research utilizes advanced imaging methods in cryo-electron microscopy and image analysis together with other complementary biophysical techniques to delineate molecular-level understanding of systems that are important targets for biotherapeutics.
 

Anti-feeding prophage -A bacterial nano-microinjection device

new_fig1_400pxWe are elucidating the functional mechanism of a nano microinjection device, Afp (anti-feeding Prophage). Afp is a complex of 18 different protein, evolved by bacteria Serratia entomopihila that delivers a toxin to kill Costelyira zealandica, commonly known as grass grub, a widespread pest of pasture that causes huge economic loss to New Zealand. Figure shows 20A resolution domain level architecture of the nanodevice as well as its toxin cargo encased in the central sheath. With high-resolution data from the newly acquired TF20 microscope we will be pushing towards atomic resolution by visualizing images of Afp in its various conformational snapshots to reveal functional insight into this protein secretion system. This detailed understanding of such a mobile protein delivery system and its unusual eukaryotic target specificity could guide rational designing of nanoscale devices and hence open cutting edge therapeutic avenues such as delivering antigenic proteins in anti-tumor immunotherapy. In collaboration with Dr. Mark Hurst of AgResearch and supported by Marsden Fund.

1) Heymann, J. B., Bartho, J. D., Rybakova, D., Venugopal, H. P. et al. (2013) 3-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila. J. Biol. Chem. 288: 25276-25284.

2) Rybakova, D., Radjainia, M., Turner, A., et al. (2013). Role of antifeeding prophage (Afp) protein Afp16 in terminating the length of the Afp tailocin and stabilizing its sheath. Mol. Microbiol. 89: 702-714.Rybakova, R.

 

Adiponectin - Assembly and engineering of peptide based therapeutic

Adiponectin_cover_300pxObesity and associated metabolic disorders are gradually becoming a pandemic internationally. Adiponectin is a protein hormone secreted by adipose tissue with remarkable remedial activities against most obesity-related disorders. Adiponectin has a globular C-terminal domain similar to complement factor 1q and TNF, a collagen-like domain and a N-terminal variable domain (AHD). These domains constitute the trimeric low molecular weight (LMW) adiponectin building block, which can oligomerize through disulphide bridges via a conserved cysteine (C39) in AHD to form the hexameric medium molecular weight (MMW) and 12-18meric high molecular weight (HMW) complexes that endow adiponectin with insulin-sensitizing activity. We have visualized the polymorphic nature of the HMW. We have also shown that the conserved W42 in AHD regulates the HMW formation. Currently we are investigating the role of other key residues in AHD and that of ER chaperones to reveal a comprehensive molecular-level understanding of adiponectin oligomerization and discover various agents that alleviate obesity-related diseases. In collaboration with Dr. Yu Wang University of HongKong and supported HRC grant.

1) Radjainia, M., Huang, B., Bai, B., et al. (2012) A highly conserved tryptophan in the N-terminal variable domain regulates rate of disulfide bond formation and oligomeric assembly of adiponectin. FEBS J. 279: 2495-2507.

2) Radjainia, M., Wang, Y., and Mitra, A.K (2008) Structural polymorphism of oligomeric adiponectin visualized by electron microscopy. J. Mol. Biol. 381:419-430.

 

Insights into poxvirus assembly

 

new_fig_3Poxviruses are the largest viruses infecting humans and among the most complex members of the virus world. Early steps in poxvirus assembly leading to the formation of obligatory immature virions (IV) have generated particular interest because of the atypical crescent-shaped precursors of IV. The role of a scaffolding protein called D13 has emerged as central. This protein has long been known to be the target of rifampicin that reversibly blocks viral assembly but the mechanism of inhibition is unknown. The primary role of D13 is to form a honeycomb scaffold (see figure) on the surface of IV and crescents. We have produced in vitro lipid vesicles that remarkably mimic the IV (see figure) both in having similar dimension (~300nm) as also having the surface honeycombed lattice of D13 as in vivo. new_fig_4We are applying electron tomography and electron crystallography for a) molecular understanding of IV formation b) design of inhibitors for D13 association on IV and c) dissection of the IV protein interactome comprised of six other proteins apart from D13. Investigating the molecular assembly of IV is a powerful probe to understand ancestral and fundamental processes of the infectious cycle of a whole class of lipid-containing viruses. In collaboration with Dr. F. Coulibaly at Monash University.

1) Hyun, J.K., Accurso, C., et al. (2011) Membrane remodeling by the double-barrel scaffolding protein of poxvirus. PLoS Pathogens. 7(9): e1002239.

2) Hyun, J-K, Coulibaly, F., Turner, A. P. et al. (2007) The structure of a putative scaffolding protein of immature poxvirus particles as determined by electron microscopy suggests similarity with capsid proteins of large icosahedral DNA viruses. Jour. Virol. 81:11075-11083.

 

 

Ongoing Research

Protein toxins

Colicin Ia

new_fig_5Colicins are a family of soluble protein toxins that kill sensitive E.coli strains, by parasitizing outer membrane receptor to gain access. Colicin Ia is a harpoon shaped a-helix rich 72kD protein consisting of receptor binding, translocating and channel forming domains linked by very long a-heices. The channel forming domain, which bears structural homology with similar putative domains in diphtheria toxin, BcL family of apoptotic proteins forms a voltage-gated non-specific ion channel in the plasma membrane to deplete the cell of essential nutrients. We are engaged in imaging the colicin Ia channel in its functional lipid-integrated state. Towards this end we have generated 2-D crystals of colicin Ia in lipid bilayer that reveals a vestibular architecture of an oligomeric channel at modest resolution and indicates a profound conformational change in the extrabilayer portion of the protein (see figure). new_fig_6Soluble form of the membrane -insertable state has also been generated that are under current, high-resolution structural study. This study will reveal details of the conformational transition shedding light on the channel architecture.

1) Greig, S. L., Radjainia, M., and Mitra, A. K. (2009). Oligomeric structure of colicin Ia channel in lipid bilayer membranes. J. Biol. Chem. 284:16126-16134.

 

 

 

 

 

Anthrax Toxin

new_fig_7_300pxAnthrax is an infectious disease that primarily affects grazing animals that come in contact with highly resilient, soil-resident spores of the gram-positive, rod-shaped and facultative anaerobic bacterium Bacillus anthracis. Anthrax toxin (AT), a cocktail of three proteins, acts through two exotoxins, lethal toxin (LeTx) and edema toxin (EdTx). These are produced when the third protein PA63 is protectolytically cleaved at the host-cell surface to produce heptamers (prepores). The toxic complex upon endocytosis forms channels in the late endosomal membrane to allow passage of the toxic moieties to the cytosol. Various neutralizing monoclonal antibodies are generated to combat anthrax. We discovered that the highly potent neutralizing mAb 1G3 is able to rearrange the quaternary organization and distort the oligomeric state of intact heptameric prepores (see figure). new_fig_8_300pxOur results from 3-D reconstruction suggest that two 1G3 molecules can distort two heptamers and produce what appear to be neutralized dodecameric PA63 complexes. Preincubating the prepores with LF does not protect them from reorganization by 1G3. This economy in function is remarkable considering that one antibody is sufficient to irreversibly compromise the structural integrity of one heptamer that is indicated to have three LFN binding sites. We are interested in delineating at high resolution the structure of this supercomplex as also the structure of the heptamer after conversion into pores. This work can set the stage of screening for effective vaccines that instigate similar structural alteration in the prepore structure. In collaboration with Dr. S. Leppla at NIH, USA.

1) Radjainia, M., Hyun, J-K., Leysath, C. E. et al. (2010) An anthrax toxin-neutralizing antibody reconfigures the protective antigen heptamer into a supercomplex. Proc. Natl. Acad. Sci. USA. 107:14070-14074.

2) Tama, F., Ren, G., Brooks III, C. L. and Mitra, A. K. (2006) Model of the toxic complex of anthrax: responsive conformational change in both the protective antigen heptamer and the lethal factor upon complexation. Prot. Sci. 15:2190-2200.

 

Membrane proteins

Drug efflux complex

The renewal of outbreaks of tuberculosis and cholera and the pronounced resistance of these, and other common pathogenic bacteria towards most antibiotics has posed a serious threat both in the developed and developing countries. We have chosen the plasma membrane integral membrane protein AcrB of the AcrA/AcrB/TolC drug-efflux complex in Escherichia coli as a model system to gain insight into the molecular mechanism of multi-drug resistance. We have generated reproducibly 2D crystals of AcrB in the natural lipid habitat, that show that AcrB can pack in many different ways, not seen in 3D crystals that were devoid lipid> this polymorphism in packing may have an important role in AcrB function. We have proceeded towards optimizing conditions for generating the elusive tripartite complex, the work on which is still undergoing. The clinical and medical relevance of the project is extended also to mammalian AcrB homologues e.g. the Niemann-Pick transporter type C1, genetic defect of which leads to aberrant regulation of cellular cholesterol homeostasis and the hedgehog receptor Patched involved in hedgehog signaling pathway whose dysfunction is implicated in tumor initiation and growth.

Aquaporin water channel

new_fig_9Aquaporins are membrane channel proteins important for maintaining fluid balance. Fluid imbalances can lead to numerous pathological conditions e.g. glaucoma, brain oedema, congestive heart failure, stroke and obesity. Trangenic knockout mice lacking the gene for AQP1 display reduced osmotic water permeability in the choroid plexus, lower intracranial pressure and intraocular pressure while those lacking AQP4 have reduced oedema upon brain injury. Our research is focused upon aquaporin 1 and 4, which facilitating rapid and specific transport of water, are drug targets for controlling fluid-related disorders. Starting with lead compounds, we propose to develop inhibitors for aquaporin 1 optimized by iterative structure-based programme, chemical synthesis and supported by in vitro assay of water transport inhibition. We also propose to initiate a similar programme of discovering inhibitors of aquaporin 4 with the long-term goal of controlling the often, fatal brain oedema caused by severe injury.

1) Ren, G., Reddy, V. S., Cheng, A., et al. (2001). Visualization of a water-selective pore by electron crystallography in vitreous ice. Proc. Natl. Acad. Sci. USA. 98:1398-1403.

 

EM facilities and accessories

TEM

Tecnai FEG20 200kV cryo-TEM is specified with STEM and HAADF imaging modes, GATAN GIF energy filter and Edax EDS. Gatan Ultrascan 1000 4 Mpixel digital camera. FEI Tomography software is provided.

Tecnai 12 120 kV cryo-TEM is equipped with a Lab6 gun and a GATAN Ultrascan 1000 4 Mpixel digital camera.

CM12 120 kV screening TEM is equipped with single tilt, rotational, double tilt, and multiple specimen holders, and a GATAN Bioscan 1 Mpixel digital camera for widefield imaging.

Specimen holders

Two GATAN 626 (60 deg tilt), one GATAN 626 (70 deg tilt), One GATAN 914 (80 deg tilt), one double-tilt holder (FEI type FP6595/20). Single tilt room temperature tomography holders FEI type and one Fischione type 2020.

Cryo-specimen preparation

1 Mark IV FEI Vitrobot, 2 manual guillotine type freezing device.

Optical difractometer

An in-house manufactured laser diffractometer with video image capture for screening of low and high-resolution TEM images.

For further details of available facilities see http://www.bioscienceresearch.co.nz/services/1067/transmission-electron-microscopy/

Structures listed in Database/structure gallery/cover art

A) AQP1 3.7A structure Protein data bank (PDB) deposition 1IH5; RSV CA T=1 icosahedron reconstruction EM data Bank entry EMD-1710; Afp 3-D reconstruction EM data Bank entry EMD-2419.

B) Facebook page of PDB in Europe (PDBe) August 9, 2013 EMD-2419 entry.

C) Cover Art
Mol. Microbiol. Volume 89(4) August 2013; Structure Volume 17(5) May 2009; Journal of Molecular Biology Volume 417(3) March 2012; Journal of Biological Chemistry Volume 284 (24) June 12 2009; Journal of Molecular Biology Volume 381(2) August 29 2008; Protein Science Volume 15 (9) September 2006.

Other research initiatives

1. In collaboration with Prof. Juliet Gerrard we are revealing high-resolution structures of novel high-molecular weight assemblies of clinically important human protein peroxiredoxin that have potential applications in nanotechnology.

2. In collaboration with Prof. Richard Cannon of University of Otago and funded by a Marsden grant we are involved in high-resolution structural studies of fungal, multi-drug efflux ABC protein Cdr1p.

Positions available

1. A Marsden funded postdoctoral fellow (21/2 yr.) is available from March 2014. The selected person will have noted strength in macromolecular microscopy and image analysis to study high-resolution structure of Afp, its different configurational states and functionally modified mutants using cryo-electron microscopy.

2. A 3-year Marsden funded PhD position is available from March 2014. The selected candidate will have strong molecular biology skills and will be involved in a study of designed Afp mutants and assessment of function associated with Afp component proteins. This student will be based at Ag Research, Lincoln in the laboratory of Dr. Mark Hurst.

3. Masters and Honours positions available based on the projects described above.

Current Lab personnel

Dr. Mazdak Radjainia - Postdoctoral Research Fellow
Lutz Hampe – PhD student
Daria Rybakova – PhD student (with Ag Research)
Eddy Chung – Masters student
Ben Rushton – Masters student
Hariprasad Venugopal – Research Technician, Computer Manager
Kien Ly- Research Technician (part time)

Past Members

Dr. Liam O’Ryan – Postdoctoral Research Fellow (2011-2013)
Sanketh Gupta – Masters student (2012-2013)
Dr. Anindito Sen – Postdoctoral Research Fellow (2009-2011)
Damon Colbert – Research Technician (2007-2010)
Dr. Jae-Kyung Hyun – Masters Student (2005), PhD student (2006-2010)
Dr. Mazdak Radjainia – PhD student (2005-2009)
Joseph D. Bartho – Research Technician (2011-2012)
Dr. Zdenko Gardian – Postdoctoral Research Fellow (2010)
Dr. Sarah Greig – Postdoctoral Research Fellow (2004-2008)
Dr. Peter Brown – Postdoctoral Research Fellow (2007)
Suzanne Manning – Research Technician (2003 – 2007)
M. Rost – International M. Diplom. student (2007)
K. N Goldie – PhD student, EMBL, main supervisor (2004-2009)
Irene Liang – Research Technician (2007-2009)
Kien Ly – Research Technician (2009-2010)
Jingyi Li – Research Technician (2004-2006)
Srdjan Mitrovic – Research Technician (2003-2004)
Connor Wright – Research Technician (2004-2005)
Dr. Alexandrine Froger – Postdoctoral Research Fellow (2002-2004)

At Scripps Research Institute, La Jolla, CA, USA
Anchi Cheng – Postdoctoral Fellow (1995-1997)
Gang Ren – Postdoctoral Research Fellow (1997-2002)
Joel Quispe – Research Technician (2000-2002)
Alexandrine Froger – Postdoctoral Research Fellow (2000-2002)
Corey Atteridge – Research Technician (1999-2000)
Peter Melnyk – Research Technician (1996-1999)

Postgraduate supervision

Research projects at the undergraduate and postgraduate levels are available immediately. Please also inquire about PhD thesis projects.

Contact: a.mitra@auckland.ac.nz

Areas of expertise

Structural Biology

Selected publications and creative works (Research Outputs)

  • De Leon-Rodriguez, L. M., Hemar, Y., Mitra, A. K., & Brimble, M. A. (2017). Understanding the metal mediated assembly and hydrogel formation of a β-hairpin peptide. Biomaterials science10.1039/c7bm00512a
    Other University of Auckland co-authors: Luis De Leon Rodriguez, Margaret Brimble, Yacine Hemar
  • Jaballah, S. A., Bailey, G. D., Desfosses, A., Hyun, J., Mitra, A. K., & Kingston, R. L. (2017). In vitro assembly of the Rous Sarcoma Virus capsid protein into hexamer tubes at physiological temperature. Scientific Reports, 7 (1)10.1038/s41598-017-02060-0
    URL: http://hdl.handle.net/2292/36114
    Other University of Auckland co-authors: Richard Kingston
  • Jeon, J., Qiao, X., Hung, I., Mitra, A. K., Desfosses, A., Huang, D., ... Gan, Z. (2017). Structural Model of the Tubular Assembly of the Rous Sarcoma Virus Capsid Protein. Journal of the American Chemical Society, 139 (5), 2006-2013. 10.1021/jacs.6b11939
    Other University of Auckland co-authors: Richard Kingston
  • De Leon-Rodriguez, L. M., Hemar, Y., Mo, G., Mitra, A. K., Cornish, J., & Brimble, M. A. (2017). Multifunctional thermoresponsive designer peptide hydrogels. Acta biomaterialia, 47, 40-49. 10.1016/j.actbio.2016.10.014
    URL: http://hdl.handle.net/2292/31304
    Other University of Auckland co-authors: Yacine Hemar, Jillian Cornish, Margaret Brimble, Luis De Leon Rodriguez
  • Yewdall, N. A., Venugopal, H., Desfosses, A., Abrishami, V., Yosaatmadja, Y., Hampton, M. B., ... Radjainia, M. (2016). Structures of Human Peroxiredoxin 3 Suggest Self-Chaperoning Assembly that Maintains Catalytic State. Structure (London, England : 1993), 24 (7), 1120-1129. 10.1016/j.str.2016.04.013
    Other University of Auckland co-authors: Juliet Gerrard, David Goldstone, Yuliana Yosaatmadja
  • Patil, R. V., Xu, S., van Hoek, A. N., Rusinko, A., Feng, Z., May, J., ... Irigoyen, M. (2016). Rapid Identification of Novel Inhibitors of the Human Aquaporin-1 Water Channel. Chemical Biology & Drug Design, 87 (5), 794-805. 10.1111/cbdd.12713
  • De Leon-Rodriguez, L. M., Kamalov, M., Hemar, Y., Mitra, A. K., Castelletto, V., Hermida-Merino, D., ... Brimble, M. A. (2016). A peptide hydrogel derived from a fragment of human cardiac troponin C. Chemical communications (Cambridge, England), 52 (21), 4056-4059. 10.1039/c6cc00209a
    Other University of Auckland co-authors: Yacine Hemar, Margaret Brimble, Luis De Leon Rodriguez
  • Khoshouei, M., Radjainia, M., Phillips, A. J., Gerrard, J. A., Mitra, A. K., Plitzko, J. M., ... Danev, R. (2016). Volta phase plate cryo-EM of the small protein complex Prx3. Nature communications, 710.1038/ncomms10534
    Other University of Auckland co-authors: Juliet Gerrard