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

Abstract

Prokaryotic cells lack a proper dedicated nuclear arrangement machinery. A set of proteins known as nucleoid associated proteins (NAPs) perform opening and closure of nucleic acids, behest cellular requirement. Among these, a special class of proteins analogous to eukaryotic histones popularly known as histone-like (HU) DNA binding proteins facilitate the nucleic acid folding/compaction thereby regulating gene architecture and gene regulation. DNA compaction and DNA protection in Helicobacter pylori is performed by HU protein (Hup). To dissect and galvanize the role of proline residue in the binding of Hup with DNA, the structure-dynamics-functional relationship of Hup-P64A variant was analyzed. NMR and biophysical studies evidenced that Hup-P64A protein attenuated DNA-binding and induced structural/stability changes in the DNA binding domain (DBD). Moreover, molecular dynamics simulations and ¹⁵N relaxation studies established the reduced conformational dynamics of P64A protein. This comprehensive study dissected the exclusive role of evolutionarily conserved apical proline residue in regulating the structure and DNA binding of Hup protein as P64 is presumed to be involved in the external leverage mechanism responsible for DNA bending and packaging, as proline rings wedge into the DNA backbone through intercalation besides their significant role in DNA binding.

Figure 1

Structural features of HU family proteins. (A) Monomeric subunit showing secondary structural elements; Initial two α-helices (α1, and α2) and α3 helix at end interspersed with β1-β5 strands. (B) Dimeric conformation of HU protein comprising of DNA binding pocket, DNA binding domain (DBD) and Dimerization domain (DD). (C) Overlaid structure of HU homologues from Mycobacterium tuberculosis (green, PDB ID: 4PT4), Mycoplasma gallisepticum (peach, PDB ID: 2NDP), and Geobacillus stearothermophilus (purple, PDB ID: 1HUE) with their conserved apical proline residue represented as sphere of respective color. Structure of HU protein of Anabaena bound to DNA (PDB ID: 1P78): (D) lateral view and (E) top view showing interaction/intercalation of proline residues. The graphical structures were generated using PYMOL software.

Figure 2

Biophysical characterization and DNA binding assay of Hup proteins (WT and P64A): (A) SEC profile of Hup proteins (WT, blue and P64A, red) compared with that of chymotrypsin (purple line, MW 25 kD) and pepsin (green line, MW 34.5 kDa) as standard reference proteins. (B) CD spectroscopy profile of Hup proteins (WT, blue and P64A, red) depicting the secondary structural characteristics. (C) Interactions between DNA and Hup proteins (WT/P64A) observed by agarose gel electrophoresis showing hp-DNA (25 bases, lane 1), WT:hp-DNA complex (lane 3), and P64A:hp-DNA complex (lane 5). Fluorescence quenching experiments showing a gradual decrease in fluorescence from WT protein (D) and P64A protein (E) after sequential addition of hp-DNA to obtain Hup:hp-DNA complex in molar ratio ranging 1:0.1 to 1:5. (F) Double logarthimic plots depiciting the dissociation constants (K_d) values for interaction of WT:hp-DNA (blue) and P64A:hp-DNA (red).

Figure 3

Backbone resonance assignment of Hup-P64A protein using NMR spectroscopy: (A) 2D- ¹H–¹⁵N HSQC spectra of Hup-P64A mutant with annotated backbone amide signals. The residues belonging to the dimeric (D) conformation and the monomeric (M) conformation are marked with blue and red color, respectively. (B) Primary sequence of protein showing the assigned monomeric (M) residues, proline residues and mutated residue (marked with an asterisk, *) are highlighted in red, cyan and green, respectively. (C) Residues present in both dimeric and monomeric conformation are represented as red spheres on a monomer subunit of three-dimensional structure of Hup dimer generated by PYMOL software. The mutated residue A64 (P64A) is represented as green sphere. (D) Residue wise intensity ratio of dimer and monomer conformation of Hup proteins (WT, blue and P64A, red).

Figure 4

Secondary structural preferences of Hup proteins (WT and P64A) estimated using NMR spectroscopy. Residue-wise comparison of cumulative secondary chemical shifts indices (Δδ^CUM) of Hup proteins (WT, blue bar and P64A, red spheres): (A) dimeric conformation; (B) monomeric conformation. The secondary structure preferences for P64A protein are shown at the top as an arrangement of α-helix (purple bar), and β-strand (cyan arrow) with mutated P64 residue (marked with an asterisk, *).

Figure 5

Comparative chemical shift analysis of Hup proteins (WT and P64A). (A) Selective overlay of ¹H–¹⁵N HSQC spectra of Hup proteins (WT, blue and P64A, red) showing peak shifts. (B) Chemical shift perturbations observed in the P64A protein dimer (blue bar) and monomer (red dots) due to the P64A mutation in the Hup protein. The cutoff value of chemical shift was decided on the basis of average chemical shift perturbation value and is denoted by black dotted line (∼0.8 ppm).The secondary structure preferences for P64A protein are shown at the top as an arrangement of α-helix (purple bar), and β-strand (cyan arrow) with mutated P64 residue (marked with an asterisk, *). (C) Residues showing significant chemical shift perturbations greater than average cutoff value are represented as spheres (Dimer, blue, and monomer,red) on different monomeric subunit of three-dimensional structure of Hup dimer generated by PYMOL software. The mutated residue A64 (P64A) is represented as green sphere.

Figure 6

Temperature dependent structural changes in Hup proteins (WT and P64A). Residue-wise temperature coefficients of (A) WT protein dimer (blue) and monomer (red) (B) P64A protein dimer (blue bar) and monomer (red dots). The secondary structure preferences for P64A protein are shown at the top as an arrangement of α-helix (purple bar) and β-strand (cyan arrow) with a mutated P64 residue (marked with an asterisk, *). Correlation map between temperature coefficients of Hup proteins (WT and P64A): (C) dimer and (D) monomer. (E) Residues showing the deviation of >2 ppb/K from the diagonal are represented as spheres (dimer, blue; monomer, red) on the three-dimensional structure of Hup dimer generated by PYMOL software. The mutated residue A64 (P64A) is represented as green sphere.

Figure 7

Stability analysis of Hup proteins (WT and P64A). ¹H–¹⁵N HSQC spectra of Hup proteins (WT and P64A) depicting H/D exchange of (A) the WT protein and (B) the P64A protein recorded for 60 min with a dead time of 10 min. (C) Protected residues common for Hup proteins (WT and P64A) are marked with a blue color while those exclusive for a protein are marked with a red color on the primary sequence of Hup proteins (WT and P64A). The secondary structure preferences for P64A protein are shown at the top as an arrangement of α-helix (purple bar), and β-strand (cyan arrow) with mutated P64 residue (marked with an asterisk, *). Protected residues showing peaks in ¹H–¹⁵N HSQC spectrum after 60 min have been marked on three-dimensional structure of Hup protein (D, WT and E, P64A). Protected residues common for Hup proteins (WT and P64A) are represented using blue spheres while those exclusive for a particular variant are represented using red spheres on one of the monomeric subunits of the Hup protein.

Figure 8

Conformational dynamics of Hup proteins (WT and P64A). Overlay of structural ensembles of Hup proteins obtained through MD simulation: (A) WT and (B) P64A showed differences in the β-arm region at various time intervals of the trajectory [0 ns, peach; 100 ns, green; 200 ns, purple; 300 ns, yellow; 400 ns, pink; and 500 ns, cyan]. Graphs representing the variation in RMSD over the time (C), and root-mean-square fluctuation (RMSF) (D) for each residue of the Hup (WT/P64A) proteins.

Figure 9

NMR-based ¹⁵N relaxation analysis of Hup-P64A protein. Residue-wise overlay of longitudinal relaxation rates (R₁) (A), transverse relaxation rates (R₂) (B), and steady state Het-NOE (C), observed for the P64A protein (dimer, blue bar, and monomer, red dots). The transverse relaxation (R₂) difference value of Hup proteins (WT and P64A) (D), calculated for each residue. The secondary structure preferences for P64A protein are shown at the top as an arrangement of α-helix (purple bar), and β-strand (cyan arrow) with mutated P64 residue (marked with an asterisk, *). (E) Residues showing significant differential transverse relaxation, i.e., above the chosen cutoff value, represented as spheres (blue) on a monomer subunit of three-dimensional structure of Hup dimer generated by PYMOL software. The mutated residue A64 (P64A) is represented as green sphere.

Figure 10

Summary of the residues exhibiting altered structural/stability/dynamics features in the DNA binding domain of Hup-P64A variant. (A) Residue-wise representation of perturbed residues in the β-arm region as determined using CSP, temperature coefficients, hydrogen exchange and relaxation analysis. (B) Residues showing perturbation shown as spheres on the three-dimensional structure of Hup dimer generated by PYMOL software. The mutated residue A64 (P64A) is represented as green sphere. The residues showing significant differences in one of the NMR parameter (CSP/temperature coefficient/hydrogen–deuterium (H/D) exchange/¹⁵N relaxation) are represented with red color, whereas residues showing differences in more than one parameter, i.e., two or more are highlighted with blue color on both the sequence and the structure.