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

Abstract

Here we report bisphenol derivatives of fluorene (BDFs) as a new type of chemical probes targeting a histone-like HU protein, a global regulator of bacterial nucleoids, via its dimerization interface perturbation. BDFs were identified by virtual screening and molecular docking that targeted the core of DNA-binding β-saddle-like domain of the HU protein from Spiroplasma melliferum. However, NMR spectroscopy, complemented with molecular dynamics and site-directed mutagenesis, indicated that the actual site of the inhibitors’ intervention consists of residues from the α-helical domain of one monomer and the side portion of the DNA-binding domain of another monomer. BDFs inhibited DNA-binding properties of HU proteins from mycoplasmas S. melliferum, Mycoplasma gallicepticum and Escherichia coli with half-maximum inhibitory concentrations in the range between 5 and 10 µM. In addition, BDFs demonstrated antimicrobial activity against mycoplasma species, but not against E. coli, which is consistent with the compensatory role of other nucleoid-associated proteins in the higher bacteria. Further evaluation of antimicrobial effects of BDFs against various bacteria and viruses will reveal their pharmacological potential, and the allosteric inhibition mode reported here, which avoids direct competition for the binding site with DNA, should be considered in the development of small molecule inhibitors of nucleoid-associated proteins as well as other types of DNA-binding multimeric proteins.

Acetylation at Lysine 86 of Escherichia coli HUβ Modulates the DNA-Binding Capability of the Protein

“…Nevertheless, a DNA-bound structure of Anabaena HU (pdb: 1P51; Swinger et al, 2003), which shares 42% sequence identity and 71% sequence similarity to E. coli HUβ, is available and was employed to generate a homology model using SWISS-MODEL (Biasini et al, 2014;Bienert et al, 2016). Based on the homology model, the β-ribbon arms (amino acid residues 56-73) and the C-terminal α-helix (amino acid residues 82-90) are involved in DNA binding (Figure 1), and this assertion is supported by previous biochemical studies of E. coli HU (Bhowmick et al, 2014;Hammel et al, 2016;Agapova et al, 2020;Thakur et al, 2021). A report of E. coli acetylome revealed acetylation at Lys3/9/18/67/86 of HUβ (Castaño-Cerezo et al, 2014).…”

Bacterial nucleoid-associated protein HU as an extracellular player in host-pathogen interaction

“…Effects of various chemical compounds on the HU protein function were studied. Among them, stilbene derivatives that inhibit HU protein binding to DNA in M. tuberculosis ( Bhowmick et al., 2014 ) and HU homologue in African swine fever virus (ASFV) that lead to the decreased ability of replication in macrophages ( Liu et al., 2020 ), bisphenol derivatives of fluorene (BDFs) that decrease DNA-binding capacity of the HU protein in mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum; and E. coli ( Agapova et al., 2020a ; Agapova et al., 2020b ). Other chemical compounds that could have negative effects on the stability of monomers of the S. melliferum HU protein and thus abolish the protein function were searched using virtual screening ( Talyzina et al., 2017 ; Agapova et al., 2019 ; Agapova et al., 2020 ).…”

“…Among them, stilbene derivatives that inhibit HU protein binding to DNA in M. tuberculosis ( Bhowmick et al., 2014 ) and HU homologue in African swine fever virus (ASFV) that lead to the decreased ability of replication in macrophages ( Liu et al., 2020 ), bisphenol derivatives of fluorene (BDFs) that decrease DNA-binding capacity of the HU protein in mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum; and E. coli ( Agapova et al., 2020a ; Agapova et al., 2020b ). Other chemical compounds that could have negative effects on the stability of monomers of the S. melliferum HU protein and thus abolish the protein function were searched using virtual screening ( Talyzina et al., 2017 ; Agapova et al., 2019 ; Agapova et al., 2020 ). Virtual screening was used also by a group of Dey and Ramakumar (2020) , where they predict chemical substances that may have an inhibitory effect on mycobacterial HU protein, among them maltotetraose, valrubicin, iodixanol, enalkiren, indinavir, carfilzomib, oxytetracycline, quinalizarin, raltitrexed, epigallocatechin and their analogues.…”

Conserved Apical Proline Regulating the Structure and DNA Binding Properties of Helicobacter pylori Histone-like DNA Binding Protein (Hup)

“… 9 , 10 However, exceptional cases where a β-strand is present prior to α 1 helix, results in increased number of β-strands as observed in an HU homologue. 8 The dimeric structure thus formed has two functional domains i.e., dimerization domain (DD) and DNA binding domain (DBD) with their exclusive functions 7 , 11 ( Figure 1 B).…”

“…Each monomeric unit follows basic HU/IHF (integrative host factor) clade structural fold with α 1 and α 2 separated by a small loop region; β-strands (β1−β5) arranged in tandem between α 2 and α 3 . , However, exceptional cases where a β-strand is present prior to α 1 helix, results in increased number of β-strands as observed in an HU homologue . The dimeric structure thus formed has two functional domains i.e., dimerization domain (DD) and DNA binding domain (DBD) with their exclusive functions , (Figure B).…”