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

Thakur, Bhishem; Gupta, Archit; Guptasarma, Purnananda

doi:10.1016/j.bbrc.2020.11.088

Cited by 3 publications

(3 citation statements)

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“…Autonomous folding, assembly, and DNA binding by the CTD of HU chains have recently been established by us through the creation of a novel NTD-lacking DNA-binding protein, which uses two fused CTDs derived from the HU chains of two different organisms. 22 HU homodimers and heterodimers are discernible through a combination of ion-exchange chromatography and analytical electrophoretic separation of HU-A and HU-B chains, which is accomplished using AUT gels that incorporate acetic acid, urea, and Triton X-100. 5−7 It is reported that mixing of wildtype HU-AA and HU-BB homodimers (independently created through chromatographic separation of unfolded HU-A and HU-B chains, followed by refolding into folded homodimers, prior to mixing) leads to their spontaneous reorganization to form HU-AB heterodimers, to a level of ∼90% of the population.…”

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“…HU-A and HU-B possess a helix-dominated N-terminal domain (NTD) comprising residues 1–44 and a sheet/IDR-dominated, highly basic, C-terminal domain (CTD) comprising residues 45–90. Both the NTD and the CTD are engaged in intersubunit as well as intrasubunit interactions, within both homodimers and heterodimers. , Intersubunit CTD-CTD interactions are hydrophobic, involving stacking interactions between antiparallel β-strands, as seen in Figure D, with charge–charge interactions involving CTD occurring mainly with DNA, and not between (or within) subunits. Intersubunit NTD-NTD interactions involve two ionic interactions (between the α-amino group of residue, M1, and the side chain of residue, D40, and between the side chains of residues D8 and K18), in addition to hydrophobic interactions that occur between abutting pairs of helices (a short helix of nearly three turns and a long helix of five and a half turns comprising a helix-turn-helix motif).…”

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“…Intersubunit NTD-NTD interactions involve two ionic interactions (between the α-amino group of residue, M1, and the side chain of residue, D40, and between the side chains of residues D8 and K18), in addition to hydrophobic interactions that occur between abutting pairs of helices (a short helix of nearly three turns and a long helix of five and a half turns comprising a helix-turn-helix motif). Autonomous folding, assembly, and DNA binding by the CTD of HU chains have recently been established by us through the creation of a novel NTD-lacking DNA-binding protein, which uses two fused CTDs derived from the HU chains of two different organisms …”

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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

et al. 2021

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HU is a bacterial nucleoid-associated protein. Two homologues, known as HU-A, and HU-B, are found in Escherichia coli 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 in vivo, 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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N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate

et al. 2021

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

Arora

Thakur

Gupta

et al. 2021

Biochemical and Biophysical Research Communications

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N-terminal extensions strengthen hydrophobic inter-subunit interactions between HU’s C-terminal domains to frustrate heterodimer formation

Arora

Thakur

Mrigwani

et al. 2021

Preprint

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HU is a nucleoid-associated protein (NAP) that helps bacterial chromosomal DNA to remain compact. Escherichia coli contains two homologs of HU that are ~ 70 % identical: HU-A and HU-B. The early log phase, late log phase, and stationary phase of E. coli growth are reported to be dominated, respectively, by HU-AA homodimers, HU-BB homo-dimers, and HU-AB heterodimers. Here, we show that the formation of HU-AB heterodimers occurs to a much lower degree in HU chains that have a displaced N-terminus, whether through addition of an N-terminal affinity (polyhistidine) tag, or fusion of a fluorescent protein. A combination of mass spectrometry, spectroscopy, chromatography, and electrophoresis (exploring glutaraldehyde crosslinking of subunits) was used to study the mixing, co-expression, unfolding and refolding of HU-AA and HU-BB homodimers. The data suggests that, in HU polypeptides with N-terminal extension, whereas inter-subunit contacts between the alpha helical N-terminal domains (NTDs) undergo facile unfolding and dissociation, inter-subunit contacts between the beta sheet- and IDR-dominated C-terminal domains (CTDs) fail to do so, due to persistence of hydrophobic inter-subunit interactions between two beta sheets. This persistence causes HU to remain nominally dimeric even after substantive unfolding, and frustrates subunit exchange and heterodimer formation.

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

Cited by 3 publications

References 16 publications

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

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

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

N-terminal extensions strengthen hydrophobic inter-subunit interactions between HU’s C-terminal domains to frustrate heterodimer formation

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