Research summary

• My first interaction with structural biology was in 2014. A/Prof. Michael J Schnieders at University of Iowa, USA, gracefully gave me an opportunity to design an ensemble of protein structures using rotamer optimization in AMOEBA force-field. My contribution in the project was to test the dead-end elimination algorithm in the model systems of insulin (chain A) and calmodulin. I was among 30 meritorious students all over India to take part in this internship as a Khorana Research Scholar.

• Subsequently, in 2015, as an Indian Academy of Science summer research fellow, I learnt python programming to design bio-physical models of cellular growth.

• I joined the lab of A/Prof. Mehdi Mobli at the University of Queensland (UQ) in 2015 as a winter research fellow. In this short-term project, using homonuclear NMR assignments for structural elucidation, I characterized the role of the linker region in the double knot toxin, DkTx. To understand these linkers through a computational point of view, I worked with Dr. Alpesh Malde in Prof. Alan Mark’s lab at UQ to run molecular dynamics (MD) simulations using GROMACS software package. My early work in this project lead to initiation of multiple projects in the Mobli lab that have focused on the study of double knot toxins or repeat peptides.

• With a strong interest in structural biology, I joined A/Prof. Mehdi Mobli’s lab again, first as a master’s exchange student in 2016 and then as a PhD candidate in 2017. In master’s thesis research, I learnt expression and purification of disulfide rich peptides. Specifically, I developed the protocol for the expression and purification of Chlorotoxin (ClTx), a peptide obtained from the venom of the scorpion Leiurus quinquestriatus. Recently, using chemical shift mapping, ITC experiments and molecular docking using HADDOCK, I studied the structural basis for the interaction of this cancer targeting toxin with the vascular endothelia growth factor receptor Neuropilin-1 (NRP1).

• For my PhD thesis, I chose a multidisciplinary project to study the structural basis for the potent and selective inhibition of Na_V1.7 by Pn3a - a disulfide rich peptide obtained from the venom of the South American tarantula Pamphobeteus nigricolor. I also ran a bioinformatics screen for uncharacterized Na_V modulators. The HMMER screening of UNIPROT database led to the structural characterization of a Na_V1.7 modulator - Cg4a. The inhibitory potency of Cg4a was further improved using rational mutagenesis and whole-cell patch-clamp electrophysiology. Moreover, I performed NMR and ITC experiments to characterize the interactions of Pn3a with lipids in a nanodisc bilayer model system. To summarize, in the thesis project, I studied the mechanism-of-action of Pn3a that characterised the binding of peptide to lipids for increased potency at the Na_V1.7 channel. Overall, knowledge acquired from the thesis can be applied to develop pharmaceutical drug leads for targeting chronic pain.

Skills

1. Object-oriented programming and force-fields (computational protein design)

2. Molecular biology, protein expression and purification, teamwork

3. Biochemistry, protein science, drug design, collaborations, research, presentations

Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor Neuropilin-1

• Chlorotoxin (ClTx) is a 36-residue disulfide-rich peptide isolated from the venom of the scorpion Leiurus quinquestriatus. This peptide has been shown to have the remarkable property of selectively binding to brain tumours (gliomas), although there are conflicting reports regarding its direct cellular target. Recently, the vascular endothelial growth factor receptor, Neuropilin-1 (NRP1) has emerged as a potential target of the peptide. Here, we sought to characterise the details of the binding of ClTx to the b1-domain of NRP1 (NRP1-b1) using solution state nuclear magnetic resonance (NMR) spectroscopy.

• The 3D structure of the isotope labelled peptide was solved using multidimensional heteronuclear NMR spectroscopy to produce a well-resolved structural ensemble. The structure points to three putative protein-protein interaction interfaces, two basic patches (R14/K15/K23 and R25/K27/R36) and a hydrophobic patch (F6/T7/T8/H10). The NRP1-b1 binding interface of ClTx was elucidated using ¹⁵N chemical shift mapping and included the R25/K27/R36 region of the peptide. The thermodynamics of the binding was determined using isothermal titration calorimetry (ITC). In both NMR and ITC measurements the binding was shown to be competitive with a known NRP1-b1 inhibitor. Finally, combining all of this data we generate a model of the ClTx:NRP1-b1 complex.

• The data shows that the peptide binds to the same region that is used by the SARS-CoV-2 virus for cell entry, via a non-canonical binding mode. Our results show that the NRP1 binding motif can be fulfilled spatially while providing a basis for further engineering of ClTx with improved NRP1 binding for future biomedical applications.

• For more details - Gagan Sharma^#, Carolyne B. Braga^#, Kevin Chen, Xinying Jia, Venkatraman Ramanujam, Brett Collins, Roberto Rittner, Mehdi Mobli, “Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor Neuropilin-1”, Current Research in Structural Biology (2021),

DOI - https://doi.org/10.1016/j.crstbi.2021.07.003

Recombinant production, bioconjugation and membrane binding studies of Pn3a, a selective Na_V1.7 inhibitor

• Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Na_Vs) play a critical role in this process. In particular, the Na_V subtype 1.7, has emerged as a promising universal target for the treatment of pain.

• Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na_V1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development.

• We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

• For more details - Sharma, Gagan, Jennifer R. Deuis, Xinying Jia, Alexander Mueller, Irina Vetter, and Mehdi Mobli. "Recombinant production, bioconjugation and membrane binding studies of Pn3a, a selective Na_V1. 7 inhibitor." Biochemical Pharmacology (2020): 114148. DOI - https://doi.org/10.1016/j.bcp.2020.114148

Elucidating the Lipid Binding Properties of Membrane-Active Peptides Using Cyclised Nanodiscs

• The lipid composition of the cellular membrane plays an important role in a number of biological processes including the binding of membrane-active peptides. Characterization of membrane binding remains challenging, due to the technical limitations associated with the use of standard biophysical techniques and available membrane models. Here, we investigate the lipid binding properties of two membrane-active peptides, VSTx1, a well characterized ion-channel inhibitor, identified from spider venom, that preferentially binds to anionic lipid mixtures, and AA139 an antimicrobial β-hairpin peptide with uncharacterised lipid binding properties, currently in pre-clinical development.

• The lipid binding properties of these peptides are elucidated using nanodiscs formed by both linear and circularized (sortase-mediated) forms of a membrane scaffold protein (MSP1D1ΔH5). We find that nanodiscs formed by circularized MSPs—in contrast to those formed by linear MSPs—are sufficiently stable under sample conditions typically used for biophysical measurements (including lipid composition, a range of buffers, temperatures and concentrations). Using these circularized nanodiscs, we are able to extract detailed thermodynamic data using isothermal titration calorimetry (ITC) as well as atomic resolution mapping of the lipid binding interfaces of our isotope labeled peptides using solution-state, heteronuclear, nuclear magnetic resonance (NMR) spectroscopy. This represents a novel and general approach for elucidating the thermodynamics and molecular interface of membrane-active peptides toward flat lipid bilayers of variable composition.

• Our approach is validated by first determining the thermodynamic parameters and binding interface of VSTx1 toward the lipid bilayer, which shows good agreement with previous studies using lipid micelles and liposomes. The method is then applied to AA139, where the membrane binding properties are unknown. This characterization, involved solving the high-resolution structure of AA139 in solution using NMR spectroscopy and the development of a suitable expression system for isotope labeling. AA139 was found to bind exclusively to anionic membranes with moderate affinity (K_d~low μM), and was found to have a lipid binding interface involving the termini of the β-hairpin structure. The preference of AA139 for anionic lipids supports a role for membrane binding in the mode-of-action of this peptide, which is also consistent with its higher inhibitory activity against bacterial cells compared to mammalian cells. The described approach is a powerful method for investigation of the membrane binding properties of this important class of molecules.

• For more details - Zhang, Alan H., Ingrid A. Edwards, Biswa P. Mishra, Gagan Sharma, Michael D. Healy, Alysha G. Elliott, Mark AT Blaskovich et al. "Elucidating the lipid binding properties of membrane-active peptides using cyclised nanodiscs." Frontiers in chemistry 7 (2019): 238. DOI - https://doi.org/10.3389/fchem.2019.00238

A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors

• Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Na_Vs) have attracted particular attention as potential analgesic targets. Na_Vs, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na_V1.7) to controlling electrical signalling in cardiac function (Na_V1.5).

• Understanding the structural basis of Na_V function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na_V modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Na_Vs by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na_V1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na_V1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays.

• For more details - Zhang, Alan H., Gagan Sharma, Eivind AB Undheim, Xinying Jia, and Mehdi Mobli. "A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors." Neuroscience letters 679 (2018): 35-47. DOI - https://doi.org/10.1016/j.neulet.2018.04.030