A simulacrum of the heterodimer formed by HU-A and HU-B chains in enteric bacteria

One of the proteins that we currently study in the lab is a DNA-binding, nucleoid-associated protein (NAP) called HU. HU is a histone-like protein. In concert with DNA supercoiling, it bends and compacts DNA by a factor of over a thousand-fold in bacterial genomes, in a manner that is not yet fully understood. Thus, HU influences DNA replication, transcription of genes, and much else that happens within bacteria, and quite literally plays a central role in the lives of bacteria, since it its highest concentrations are in the nucleoid of the bacterial genome that lies in the most central space of any bacterium.

Following a 'curtain-raiser' paper on HU that we published several years ago, we have ended up not publishing our other work on HU until earlier this year.

However, we are now amending this situation, and publishing a series of papers on this highly interesting DNA-binding protein.

Our first paper on HU this year was about a protein called HU-SIMUL inspired by HU, which utilized two copies of the C-terminal domain of HU sourced from different bacteria, and folded to form a novel double-stranded DNA-binding protein. This paper was published in Biochemical and Biophysical Research Communications.

Our second paper on HU this year, was published last month in the Journal of Biological Chemistry. It presented the finding that HU binds to bacterial cell surface lipopolysachharide using aspects of its structure that otherwise bind to DNA. The profound implications of this finding for the generation of biofilms was discussed.

This post is about our third paper on HU this year. It has just been accepted for publication in Biochemical and Biophysical Research Communications. It presents a method for the creation and study of a simulacrum of the HU-AB heterodimer. The HU-AB heterodimer is known to dominate stationary-phase cultures of enteric bacteria. We show that the HU-AB heterodimer is many times more thermodynamically-stable than homodimers of either HU-A or HU-B. This provides a great explanation for why HU-AB heterodimers form at all within bacteria, as well as why they form spontaneously when HU-A and HU-B homodimers are added to each other in solution, yielding a ~ 90 % population of HU-AB heterodimers. The abstract of this paper is presented three paragraphs hence.

In our fourth paper on HU this year, which is forthcoming (i.e., it has been resubmitted after revision, and is on the verge of acceptance at a journal), we shall establish that the formation of the HU-AB heterodimer must occur through a hetero-tetrameric intermediate formed by the association of homodimers; an intermediate within which subunits are swapped without the release of any monomers into solution. In this forthcoming paper, we have established this fact by demonstrating that the subunit interface is highly-stable in all HU dimers, and also that N-terminal modifications of HU chains end up completely halting all subunit exchange to form HU-AB heterodimers, for steric and kinetic reasons.

In our fifth paper on HU this year, which we are currently preparing, having finished a particular set of studies, we present data that we expect will underlie all future understanding of how HU plays a role in compacting DNA, well above and beyond its role in the bending of DNA. We expect to upload this on BioRxiv in a few weeks.

Meanwhile, here is the abstract of the third paper, just accepted for publication at BBRC, which this post is about.

Abstract

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.