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Chemical-Structural Biology and Design

​DESCRIPTION OF LAB RESEARCH WORK
We are investing molecular mechanisms of cell adhesion protein integrins emphasizing on leukocytes. In particular, protein-protein complexes are examined which are relevant to activation and resting of the receptors. Our works have uncovered new paradigm in integrin signalling cascades.
Our laboratory is working on de novo designing mini-proteins and antimicrobial peptides. We have designed novel b-sheet proteins that bind multi-heme with affinity close to naturally occurring large proteins. A significant thrust in the face of antibiotic resistance is to develop antimicrobial peptides and understand molecular mechanisms. We are investigating lead short peptides with potent activity against drug-resistant bacteria. The work aims to explore multifunctional of b-boomerang peptides which are designed by us.

LAB MEMBERS
LEAD PI
Surajit Bhattacharyya
Associate Professor

Email: surajit@ntu.edu.sg
Phone: (65) 6316 7997
Office: SBS-02S-78
Vidhya Bharathi Dhanabal
Research Associate

Email: vbdhanabal@ntu.edu.sg


CURRENT PROJECTS
  1. Design and mode of action of antimicrobials (peptides and mimetics) and mechanisms of bacterial resistance
    Background
    Drug resistant bacterial pathogens are of significant threat to the public health around the globe. There is an urgent need to develop new antibiotics; however, the pipeline for producing new drugs has been highly reduced over past 30 years. The US Food and Drug Adminis¬tration (FDA) had approved 20 new antibiotics between 1980 and 1984, but only three new antibiotics were approved in recent years. The lack of new antibiotics is a reflection of reduced productivity of drugs in the pharmaceutical industry. As existing drugs are becoming old, finding new drugs turns out to be difficult. Most importantly, the growing number of resistant bacterial strains indicates that new antibiotics should function with a different mode of action. It is now well documented methicillin resistant Gram-positive Staphylococcus aureus (MRSA) infections are difficult to treat. Infectious diseases caused by Gram-negative bacteria are even more major threat in human health. Notably, the spread of multidrug-resistant so called ‘ESKAPE’ pathognes i.e. Enterococcus, Staphylococcus aureus, Klebsiella, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter, is an enormous challenge. Many drugs against Gram negative bacteria show limited efficacy due to the outer membrane barrier. In order to treat bacterial infections, development of novel antibiotics with different mode of action is highly critical. Cationic Antimicrobial Peptides (AMPs) are vital components of innate immunity of host defence system. AMPs are multifunctional molecules demonstrating direct killing of broad range of bacteria including multiple drug resistance strains. AMPs are found to demonstrate anti-viral, anti-fungal and anti-parasitic activity. Some AMPs have been known to kill cancer cells. Reports have also suggested signalling functions of AMPs that would modulate functions of innate immune cells. As a mode of action, most AMPs cause lysis of bacterial cells by destabilizing integrity of membranes. AMPs remain bactericidal over the course of evolution plausibly indicating difficulty of bacteria to change the membrane compositions and structures. Now, it has been well conceived that the broad spectrum antibacterial activity in conjunction with shorter size and cell selectivity of AMPs could be employed to develop novel antibiotics. Pronounced interests have been noted for structure-activity (SAR) correlations of AMPs, designing novel AMPs and various antimicrobial applications of AMPs containing organic scaffolds.
    Proposed work
    Mechanism of action of AMPs in bacterial cell killing remains unclear, due to the fact that 3-D structures of AMPs have not been obtained in appropriate cellular environments. Interactions with outer-membrane components would like to influence mode of action and mechanisms of AMPs. Bacterial cells are protected from antibacterial substances employing additional membrane components exposed to the external environment. Gram-positive bacteria contain a thick peptidoglycan layer whereas Gram-negative bacteria are surrounded by an asymmetric outer-membrane. The outer leaflet of the outer-membrane is predominantly consisted of a specialized lipid called lipopolysaccharide (LPS). By contrast to peptidoglycan, LPS establishes a permeability barrier limiting access to antibiotics, antibacterial drugs and other molecules. As LPS in the outer-membrane protects live bacteria, LPS from dead bacteria is highly toxic to humans and other animals. LPS, as known as endotoxin, is a leading agent of septic shock or sepsis. In the absence of any therapeutic modality, annually 120,000 people are estimated to be deceased due to the septic shock syndromes. There have been constant searches for effective drugs to prevent sepsis related fatality. Molecules that would bind and neutralize endotoxin are highly sort after. LPS of the outer membrane of Gram negative bacteria is critically involved in interactions with cationic AMPs. In this on-going research, we will determine 3-D structures of host defence peptides and proteins and designed peptides in LPS and correlate their activity. A broad range of methods e.g. peptide design, expression, NMR, ITC, optical spectroscopy, dynamic light scattering, biological assays are used to gain insights into structure and functions of AMPs.
    Over past and current years, my research group has determined atomic-resolution structures and mapped interactions of a number of potent AMPs with LPS by use of NMR spectroscopy. We have solved LPS-bound 3-D structures and interactions of a number of AMPs including pharmaceutically important MSI-594 (Chemistry, 2009, JACS, 2010), pardaxin (JBC, 2010), temporins (JBC, 2011, PLOS-One, 2013), beta-hairpin peptides, protegrins (BBA, 2012, BBA 2014), de-novo designed beta-boomerang AMPs (Biochemistry, 2007, JBC, 2009, Antimicrobial Agents & Chemo. 2014, Bioconjugate Chem. 2012, Chem. Comm, 2014). Our research have discovered novel structural folds of AMPs and demonstrated critical structural features of AMPs required for bacterial cell killing.

  2. Mechanisms of Integrins Mediated Cell-Cell Adhesion & Therapeutics Development
    Background
    Integrins are a large family of type I transmembrane cell adhesion molecules that are involved in many biological processes, including immunity, wound healing, and the development of metazoans. Integrins are unique signaling receptors that carry out bi-directional signaling inside-out and outside-in. Integrins mediated signaling are directly correlated with several diseases like cancer, autoimmune diseases, inflammations etc. In humans there are 24 specific integrins that can be categorized based on ligand-binding specificities or tissue expressions. Each integrin is composed of an alpha and a beta subunit that are non-covalently associated, and each subunit has a large extracellular domain that binds ligand, a transmembrane domain and a short cytoplasmic tail (CTs). The ligand-binding properties of integrins are tightly regulated by cytoplasmic proteins that interact with the integrin cytoplasmic tails. These interactions regulate the conformation of integrin by allostery that modulates its ligand-binding affinity. A large number of cytoplasmic proteins have been identified to interact directly with the integrin CTs, potentially forming multi-protein complexes. Characterization of the multi-protein complexes is deemed essential not only to understand sequence of events of complex formation but also protein/protein interface would be a viable target for therapeutic development. There is still very limited information on how multiprotein-complex involving different integrin CT-interacting partners function temporally and spatially to regulate integrin activation, and it remains to be discovered how integrin CT-interacting partners modulate the functions of one another. This is compounded by the fact that there are subtle but important differences in the sequence composition of the integrin CTs. Hence, a ubiquitous model of integrin regulation is insufficient to explain the varied signaling properties of different integrins reported in different cell types such as beta3 integrins in fibroblast compared with beta2 integrins in leukocytes.
    Proposed work
    We are investigating beta2 integrins that are only expressed in leukocytes and they are critical for a functional immune system. There are four members of the beta2 integrins: aLb2, aMb2, aXb2 and aDb2. Our current and future research aims to obtain a comprehensive understanding of the network of interactions at atomic resolution. We investigate interactions between the b2 cytosolic tail of integrins of leukocytes with its negative and positive protein regulators using NMR spectroscopy and in vivo functional analyses. Works from my laboratory have determined 3-D structures of b2-CT and a-CTs of aL (J. Biol. Chem. 2009), aM (J. Biol. Chem. 2011), aX (PLOS-One, 2012) and a4/paxillin (PLOS-One, 2013) and mapped interactions between a and b CTs of leukocytes. These results have provided important molecular insights for activation and regulation of b2 integrins and also showed critical structural and interface, a/b CTs variations with other integrins.

  3. Designing Functional Mini-proteins
    Background
    One of goals in synthetic biology is to design proteins that can mimic function of naturally occurring proteins or possess unique functions and structures. Recently, we have designed heme coordinating synthetic b-sheet mini-proteins. Heme as a protein cofactor serves a number of biological activities e.g. enzymatic, electron transfer and energy conservation; therefore, designing hemeproteins have gained considerable attention in the past and recent years. Most naturally occurring proteins bind heme with helical structures; heme binding b-sheet proteins are less frequent. Therefore, a majority of the de novo designed heme-proteins are based on helices. By contrast, assemblies of b-sheets frequently resulted in insoluble aggregations or stabilization of heterogeneous amyloid like b-structures. Consequently, de novo designing of discretely folded b-sheet proteins are often found to be challenging. The all b-sheet protein designing success remain limited to miniaturized, water soluble, b-sheet proteins containing three to four antiparallel b-strands. However, engineering biological functions or ligand binding into the miniaturized b-sheet proteins have met only limited success. In particular, b-sheet proteins demonstrating high affinity heme binding remained obscure.
    Proposed work
    Utilizing non-coded amino acids and creating heme binding pockets, we have successfully designed mini protein sequences (< 40 amino acids) that fold into desired multi-stranded b-sheet topologies and able to coordinate single heme or di-heme inside the heme binding pockets (refs: Angew Chem Int Ed Engl 2013, Chemical Science, 2016, Angew Chem Int Ed Engl 2017). In more recent on-going works (unpublished) we created b-sheet proteins that can accommodate multiple (four to eight) heme molecules. Capturing multiple heme molecules would generate novel protein based materials with enormously important applications e.g. fast enzymes, biosensors, light harvesting systems etc. In future works, we plan to develop synthetic proteins or nano-materials (conjugated with nano-particles) with heme or metal-ions based functionalities.


PUBLICATIONS
  • Interaction analyses of the integrin beta2 cytoplasmic tail with the F3 FERM domain of talin and 14-3-3zz reveal a ternary complex with phosphorylated tail. Deepak Chatterjee, Lewis Lu Zhiping, Suet-Mien Tan and Surajit Bhattacharjya* J. Mol. Biology, 2016, 428(20):4129-4142.
  • Interaction analyses of scaffold protein 14-3-3z, adaptor protein Dok1 and phosphorylated beta cytosolic tails of integrins reveal a binary switch mechanism of recognition. Deepak Chatterjee, Areetha D’Souza, Yaming Zhang, Wu Bin, Suet-Mien Tan & Surajit Bhattacharjya*, J. Mol. Biol. 2018 430(21):4419-4430.
  • Lipopolysaccharide-affinity copolymer senses the rapid motility of swarmer bacteria to trigger antimicrobial drug release. Shengtao Lu, Wuguo Bi, Quanchao Du, Sheetal Sinha, Xiangyang Wu, Arnold Subrata, Surajit Bhattacharjya, Bengang Xing & Edwin K. L. Yeow. Nature Communications 2018, 9(1):4277
  • b-hairpin peptides: heme binding, catalysis and structure in detergent micelles.
  • Mukesh Mahajan and Surajit Bhattacharjya* Angewandte Chemie (2013), 52, 6430-6434.
  • Designed heme-cage beta-sheet mini-proteins. Areetha D’Souza, Xiangyang Wu, Edwin Kok Lee Yeow, Surajit Bhattacharjya* Angew. Chem. Int. Ed. 2017, 56, 5904-5908
  • Expanding heme-protein folding space: multi-heme letcher designed b-sheet mini-proteins. Areetha D’Souza, Jaume Torres & Surajit Bhattacharjya*. Communications Chemistry-Nature 2018, 1:78 | DOI: 10.1038/s42004-018-0078-z
  • Cell selective pore forming antimicrobial peptides of the prodomain of human furin: a conserved aromatic/cationic sequence mapping, membrane disruption and atomic-resolution structure and dynamics. Sheetal Sinha, Munesh Kumar Harioudh, Rikeshwer P. Dewangan, Wun Jern Ng, Jimut Kanti Ghosh & Surajit Bhattacharjya*. ACS-Omega 2018 3, 14650-14664.
  • Resurrecting inactive antimicrobial peptides from lipopolysaccharide (LPS) trap.
  • Harini Mohanram and Surajit Bhattacharjya* Antimicrobial Agents & Chemotherapy, 2014,58,1987-1996
  • Designed b-boomerang antiendotoxic and antimicrobial peptides: structures and activities in lipopolysaccharide.
  • Anirban Bhunia, Harini Mohanram, Prerna N. Domadia, Jaume Torres, and Surajit Bhattacharjya* Journal of Biological Chemistry (2009), 284, 21991-22004. [Paper of the week].
  • NMR structure of pardaxin, a pore-forming antimicrobial peptide, in lipopolysaccharide micelles: mechanism of outer membrane permeabilization.
  • Anirban Bhunia, Prerna N Domadia, Jaume Torres, Kevin J Hallock, Ayyalusamy Ramamoorthy and Surajit Bhattacharjya* Journal of Biological chemistry (2010), 285, 3883-3895.
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