Publications by authors named "Bhishem Thakur"

4 Publications

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N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate.

Biochemistry 2021 Jun 21;60(23):1836-1852. Epub 2021 May 21.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab 140306, India.

HU is a bacterial nucleoid-associated protein. Two homologues, known as HU-A, and HU-B, are found in within which the early, late, and stationary phases of growth are dominated by HU-AA, HU-BB, and HU-AB dimers, respectively. Here, using genetic manipulation, mass spectrometry, spectroscopy, chromatography, and electrophoretic examination of glutaraldehyde-mediated cross-linking of subunits, in combination with experiments involving mixing, co-expression, unfolding, and refolding of HU chains, we show that the spontaneous formation of HU-AB heterodimers that is reported to occur upon mixing of wild-type HU-AA and HU-BB homodimers does not occur if chains possess N-terminal extensions. We show that N-terminal extensions interfere with the conversion of homodimers into heterodimers. We also show that heterodimers are readily formed at anticipated levels by chains possessing N-terminal extensions , when direct chain-chain interactions are facilitated through production of HU-A and HU-B chains from proximal genes located upon the same plasmid. From the data, two explanations emerge regarding the mechanism by which N-terminal extensions happen to adversely affect the conversion of homodimers into heterodimers. (1) The disappearance of the α-amino group at HU's N-terminus impacts the intersubunit stacking of β-sheets at HU's dimeric interface, reducing the ease with which subunits dissociate from each other. Simultaneously, (2) the presence of an N-terminal extension appears to sterically prevent the association of HU-AA and HU-BB homodimers into a critically required, heterotetrameric intermediate (within which homodimers could otherwise exchange subunits without releasing monomers into solution, by remaining physically associated with each other).
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http://dx.doi.org/10.1021/acs.biochem.1c00081DOI Listing
June 2021

HU-AB simulacrum: Fusion of HU-B and HU-A into HU-B-A, a functional analog of the Escherichia coli HU-AB heterodimer.

Biochem Biophys Res Commun 2021 Jun 5;560:27-31. Epub 2021 May 5.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India. Electronic address:

In enteric bacteria such as Escherichia coli, there are two homologs of the DNA-binding nucleoid associated protein (NAP) known as HU. The two homologs are known as HU-A and HU-B, and exist either in the form of homodimers (HU-AA, or HU-BB) or as heterodimers (HU-AB), with different propensities to form higher-order oligomers. The three different dimeric forms dominate different stages of bacterial growth, with the HU-AB heterodimer dominating cultures in the stationary phase. Due to similarities in their properties, and the facile equilibrium that exists between the dimeric forms, the dimers are difficult to purify away from each other. Although HU-AA and HU-BB can be purified through extensive ion-exchange chromatography, reestablishment of equilibrium interferes with the purification of the HU-AB heterodimer (which constitutes ∼90% of any population with equal numbers of HU-B and HU-A chains). Here, we report the creation of a functional analog of HU-AB that does not appear to partition to generate any minority populations of HU-AA or HU-BB. The analog was constructed through genetic fusion of the HU-B and HU-A chains into a single polypeptide (HU-B-A) with a glycine/serine-rich linker of 11 amino acids separating HU-B from HU-A, and a histidine tag at the N-terminus of HU-B. HU-B-A folds to bind 4-way junction DNA, and displays a significant tendency to form dimers (i.e., analogs of HU tetramers), and a higher thermodynamic stability than HU-BB or HU-AA, thus explaining why it dominates mixtures of HU-B and HU-A chains.
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http://dx.doi.org/10.1016/j.bbrc.2021.04.107DOI Listing
June 2021

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms.

J Biol Chem 2021 Mar 10:100532. Epub 2021 Mar 10.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306. Electronic address:

In biofilms, bacteria that possess a negatively-charged surface are embedded within a matrix of polymers consisting mainly of negatively-charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively-charged substance, e.g., a basic protein, exists within biofilms to neutralize charge-charge repulsions and act as a 'glue' attaching negatively-charged bacteria to negatively-charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly-abundant nucleoid-associated protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, e.g., (a) availability in association with e-DNA; (b) multivalent DNA-binding; (c) non-sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU-cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and non-canonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical-chemical, microscopy-based and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS; that this facilitates DNA-DNA, DNA-LPS and LPS-LPS interactions; and that this facilitates bacterial clumping as well as attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (non-shaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide and move away from each other, in opposition to the accreting influence of HU).
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http://dx.doi.org/10.1016/j.jbc.2021.100532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063757PMC
March 2021

A novel protein-engineered dsDNA-binding protein (HU-Simulacrum) inspired by HU, a nucleoid-associated DNABII protein.

Biochem Biophys Res Commun 2021 01 10;534:47-52. Epub 2020 Dec 10.

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab, 140306, India. Electronic address:

HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ∼44 residues and a beta strand- and IDR-rich C-terminal region (CTR) of ∼46 residues. CTR binds to DNA through (i) a clasp (two arginine/lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat surface (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.088DOI Listing
January 2021