Abhinav KV

Postdoctoral Work

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Conjugative secretion systems in gram-negative bacteria

Antibiotics have been the cornerstone of treatment for bacterial infections for many years. Unfortunately, remarkable advances in fighting bacterial infections over the years are in danger of being undone due to the rapid spread of antibiotic resistance genes among bacterial pathogens. Accounting for 700,000 deaths worldwide, the threat of antibiotic resistance presents a formidable challenge to humanity. The major mechanism by which bacteria evolve antibiotic resistance is by exchanging genetic components via horizontal gene transfer (HGT). This process of unidirectional transfer of large DNAs from a donor to a recipient cell is widespread within both gram-negative and -positive bacteria and is the major source of their genetic diversity. Responsible for almost 80% of all bacterial gene transmission involving antibiotic resistance, HGT is at the core of the current antibiotic resistance crisis. HGT can occur via a variety of mechanisms namely transformation, transduction, and conjugation. However, the main process implicated in disseminating antibiotic resistance genes in bacteria is conjugation, whereby genetic material is transferred from a donor to a recipient cell through direct contact, often mediated by a very large transmembrane machinery called “conjugative transfer machinery/system” belonging to the larger class of Type IV secretion systems (T4SSs) and a large polymer tube called the conjugation pilus. Given that secretion systems are the primary drivers of bacterial conjugation, understanding how these systems work is critical. My postdoctoral work in Prof. Gabriel Waksman's lab involved structural and biophysical characterization of two T4SSs:  from the E. coli R388 plasmid and E. coli pKM101 plasmid and a conjugative pilus from the E. coli R388 plasmid using membrane-protein biochemistry and single-particle cryo-electron microscopy (cryo-EM). My initial work on characterizing the VirB4 ATPase, a membrane associated ATPase which is the largest protein in the system as well as purifying a complex of VirB4 with another ATPase, VirB11 led to significant advances in understanding the nature of interactions that energise these machines. Most importantly, this led to the first high-resolution structure of a T4SS (Figure 1). This work (Mace and Abhinav et al., Nature, 2022) describes the first near-atomic resolution structure of a fully assembled T4SS involved in conjugation (and the first for any T4SS). The structure of this massive double membrane spanning macromolecular assembly (2.8 MegaDalton in size, containing 10 proteins and comprising over 90 polypeptides) not only elucidates only how various proteins of the T4SS come together but also illustrates an entirely novel mechanism of pilus biogenesis, completely different from the mechanism of assembly of any other known pili in bacteria. This, along with my recent work on the high-resolution structure of a detergent-free T4SS from E. coli pKM101 (unpublished) (Figure 2) and conjugative pilus from the E. coli R388 plasmid (unpublished) (Figure 3) enables us to visualise the pilus biogenesis mechanism in unprecedented detail and provides a strong framework for asking mechanistic questions about pilus biogenesis and DNA transfer. Insights from these investigations are also expected to yield targets for structure-based inhibitor design blocking conjugation and help us fight against the formidable challenge of antimicrobial-resistance (AMR). 

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PhD Work

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1. Establishing a structural basis for sugar-binding to a therapeutically important plant lectin: jacalin

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This work was aimed at undertaking structural, biophysical and computational work on a diverse class of carbohydrate binding proteins called lectins, known to be important in host-pathogen interactions, innate immunity response and plant defense (Abhinav et al., Pure and Appl. Chem., 2014). This involved resolving two specific nuances pertaining to carbohydrate binding interactions of jacalin, a plant lectin using a combination of X-ray crystallographic, in-solution experiments and modelling studies. The first objective was to probe monosaccharide anomeric selectivity, an observation with important implications in HIV therapeutics. Jacalin was first crystallized and structurally investigated in Prof. Vijayan’s lab, MBU, IISc and until very recently it was thought that beta-anomers of carbohydrates bind to jacalin with lower affinity because of the protein-induced steric clashes. However, my work, through a series of structural (X-ray crystallography) and biophysical studies demonstrated that the both alpha- and beta- anomers establish nearly the same interactions with the protein at the atomic level. However, the difference in affinity arises from the distortion in the beta-linked galactopyranose ring, both at the mono- and disaccharide levels, which results in an increase in the internal energy of the ligand, eventually leading to lower affinity than alpha-linked carbohydrates. This led to the establishment of geometrical distortion as a strategy for modulating affinity in lectins (Abhinav et al., Acta Cryst. D, 2015; Abhinav et al., IUBMB Life, 2017) (Figure 4), a completely novel concept and resulted in a paradigm shift in the field of lectin-carbohydrate interactions. The second unexplored issue addressed was to establish a relationship between the nature of the glycosidic linkage and the spatial preferences of the reducing and non-reducing ends of a disaccharide during lectin binding. The investigations led to an interesting structural rationale for this preference and a probable mode of binding for complex O-type mucins (Abhinav et al., IUBMB Life, 2016) (Figure 5). Together, these studies established a comprehensive structural basis for carbohydrate binding to lectins and has important therapeutic and diagnostic implications. 

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2. Identification of microbial (mycobacterial and archeal) lectins

Lectins have been characterized extensively from eukaryotic sources, namely plant and animals but their existence in prokaryotes remains poorly explored. This work involved carrying out exploratory bioinformatic, structural and biophysical studies on putative lectins identified using in-sillico scanning from mycobacterial (Abhinav et al., Proteins, 2013) and archeal genomes (Abhinav et al., Proteins, 2016). Some of the putative-lectins identified from the search were also cloned, over expressed, purified and biophysically characterized. Extensive structural studies on M. tuberculosis heparin binding hemagglutinin (HBHA) with complex glycosaminoglycans resulted in identification of the core disaccharide moiety required for carbohydrate binding. The β-prism I lectin from Methanoccoccus voltae, an archea, was also cloned, biophysically characterized, and crystallized. The crystal structure of this protein was determined at 2.6 Å (Sivaji & Abhinav et al., Acta F, 2017). This is the first three-dimensional structure of a lectin from the archeal domain of life (Sivaji N et al., Glycobiology. 2021) (Figure 6). 

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3. Structural basis of Quinolone synthase mediated polyketide synthesis

Quinolone synthase (AmQNS) from Aegle marmelos is a type III polyketide synthase that produces medically relevant quinolone and acridone compounds. The structural and molecular basis of its synthetic selectivity has not been demonstrated previously. This work (Mallika & Abhinav et al., BioRxiv, 2023) characterizes quinolone synthase (AmQNS) structure, a type III PKS from Aegle marmelos Correa in the native and structure-bound forms (Figure 7). It reveals that that large AmQNS’s active site entrance provided adequate space to accommodate bulky substrates with ‘cysteine-164’ in the catalytic triad (C164-H303-N336) regulates enzyme catalysis. Further, this works also establish a model framework for understanding structural limits to ketide insertion. We hypothesise that AmQNS achieves impressive synthetic diversity due to steric, electrostatic selectivity facilitating different core substrates binding. Finally, this work also demonstrates that AmQNS structurally favors ‘quinolone production’ and synthesize acridone only under significant malonyl-CoA concentrations. Resolving structural and molecular basis of AmQNS–substrate interaction associated with high selectivity and specificity is expected to aid in the development of novel antibacterials and pharmaceutical compounds.

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I also worked briefly on studying the extreme pH stress tolerance of Vibrio cholerae in human intestine and design of novel inhibitors for HIV-1 protease as part of my M. Tech and B. Tech project dissertations respectively.

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