Structural plasticity and thermal stability of the histone-like protein from Spiroplasma melliferum are due to phenylalanine insertions into the conservative scaffold

Abstract

The histone-like (HU) protein is one of the major nucleoid-associated proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter in DNA-specificity. Here, we perform an analysis of structural-dynamic properties of HU protein from Spiroplasma melliferum and compare its behavior in solution to that of another mycoplasmal HU from Mycoplasma gallisepticum. The high-resolution heteronuclear NMR spectroscopy was coupled with molecular-dynamics study and comparative analysis of thermal denaturation of both mycoplasmal HU proteins. We suggest that stacking interactions in two aromatic clusters in the HUSpm dimeric interface determine not only high thermal stability of the protein, but also its structural plasticity experimentally observed as slow conformational exchange. One of these two centers of stacking interactions is highly conserved among the known HU and IHF proteins. Second aromatic core described recently in IHFs and IHF-like proteins is considered as a discriminating feature of IHFs. We performed an electromobility shift assay to confirm high affinities of HUSpm to both normal and distorted dsDNA, which are the characteristics of HU protein. MD simulations of HUSpm with alanine mutations of the residues forming the non-conserved aromatic cluster demonstrate its role in dimer stabilization, as both partial and complete distortion of the cluster enhances local flexibility of HUSpm.

Structure-based inhibitors targeting the alpha-helical domain of the Spiroplasma melliferum histone-like HU protein

“…Earlier, we characterized the structural and motional features of HUSpm using nuclear magnetic resonance (NMR) spectroscopy; the backbone and side chain resonance assignments and the secondary structural features derived empirically from backbone chemical shifts were reported in 37 . Since NMR can also be used to study the structure–activity relationships corresponding to specific inhibitors binding to target proteins, here we performed the backbone 15 N-NMR relaxations experiments to characterize the motional features of HUSpm upon both simultaneous and separate addition of the inhibitor and DNA.…”

“…To confirm BDF4 binding to the β-saddle domain core of HUSpm, we performed titration of the 15 N-labelled protein (both in its free form and in complex with the DS14 duplex (Supplementary Table S4 )) with different amounts of the inhibitor. 15 N-HUSpm preparation and conditions of NMR spectroscopy were established in the previous work 37 . The interactions of HUSpm with the short canonical double-stranded DNA (DS14) were also subjected to the NMR study.…”

“…The heteronuclear NMR experiments 58 59 ) spectra were acquired to monitor the HUSpm interactions with DNA and BDF4. The spectra were analyzed using Computer Aided Resonance Assigment (CARA) 60 based on the previously obtained HUSpm chemical shifts 37 .…”

“…The CSPs for backbone amide 15 N 15 N H and 1 H N resonances were calculated by the formula Δδ = [(Δ 1 H N ) 2 + (Δ 15 N H /5) 2 ] 0.5 , where Δ 1 H N and Δ 15 N H are changes in the chemical shifts of backbone amide 1 H and 15 N, respectively, upon either DS14 or BDF4 binding to HUSpm, or BDF4 binding to HUSpm + DS14 complex. The CIPs for HUSpm backbone amides were calculated by the formula ΔI/I av = I/I av − I ref /I ref_av , where I and I ref were the intensities of the amide cross-peaks in the pairs of the NMR spectra of (HUSpm + DS14 and HUSpm), (HUSpm + BDF4 and HUSpm) or (HUSpm + DS14 + BDF4 and HUSpm + DS14), which were normalized by the averaged "standard" cross-peak intensity I av estimated along the region T27-K35 that undergoes minimal local flexibility of the protein backbone according to previous study 37 .…”

“…We followed the design strategy proposed in 35 for the development of chemical inhibitors of HU protein from mycoplasma Spiroplasma melliferum (HUSpm). High-resolution spatial structure of HUSpm (PDB ID 5L8Z 9 ) was used for virtual screening and docking, while its structure in solution (PDB ID 5OGU 37 ) was employed for the comparative analysis of conformational dynamics observed in the HUSpm dimer upon either independent or sequential addition of short double-stranded DNA and an inhibitor. We found out that bisphenol derivatives of fluorene (BDFs) inhibit DNA binding properties of three model HU proteins (HUSpm, HUα-homodimer from E. coli (HUEc) and HU from another mycoplasma species Mycoplasma gallisepticum (HUMgal)).…”