Characterization of a nontypeable Haemophilus influenzae thermonuclease

Abstract

Nontypeable Haemophilus influenzae (NTHi) has been shown to form biofilms, comprised of extracellular DNA (eDNA), in the middle ear and bronchus during clinical infections. Studies in our laboratory have shown that NTHi possesses a homolog of Staphylococcus aureus thermonuclease (staphylococcal thermonuclease), NTHi nuclease (NTHi Nuc, HI_1296). This enzyme had similar size, heat stability, and divalent cation requirements to those of the staphylococcal homolog as determined by light scattering and circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) analysis suggested an overall shape and substrate-binding site comparable to those of staphylococcal nuclease. However, NTHi Nuc was approximately 25-fold more active in fluorescence resonance energy transfer (FRET) activity assay than staphylococcal thermonuclease. Homology modeling implicates shorter NTHi Nuc loops near the active site for this enhanced activity.

Fig 1. Amino acid sequence of NTHi Nuc and its homology to staphylococcal thermonuclease.

Fig 1A shows the amino acid sequence of NTHi 2019 Nuc (HI_1296). The first 20 amino acids in bold are the signal sequence of the protein. Fig 1B shows the alignment of NTHi Nuc with staphylococcal thermonuclease, which has 35% conserved amino acids and Expect value (number of chance matches) of 2e-10, indicating a significant match.

Fig 2. Effect of EDTA on NTHi Nuc activity.

FRET assay shows single-stranded nuclease activity of NTHi Nuc activity. NTHi Nuc requires divalent cation as it lost activity with addition of EDTA. The respective lines are 0.03 nM NTHi Nuc alone (●), 0.03 nM NTHi Nuc and 0.3nM EDTA (■), 0.3nM EDTA alone (▲) and distilled water (▼). The experiment was repeated three times.

Fig 3. CD spectra of NTHi Nuc from 190–260 nm.

This figure shows representative curves. When NTHi Nuc was heated from 25°C (solid line) to 60°C (dotted line), the spectra was lost, indicating loss of secondary structure. When the sample was then cooled to 25°C (dashed line), NTHi Nuc underwent reversible thermal folding. Samples were referenced to buffer.

Fig 4. Effect of inhibitor on NTHi Nuc activity.

NTHi Nuc was incubated with 2 μM FRET substrate and increasing concentrations of inhibitor (pdTp). Enzyme activity was measured in FRET assay. n = 3 for 0.03nM of pdTp and n = 2 for 0.015nM of pdTp. Relative fluorescent units decreased as pdTp concentration increased, thus indicating that the inhibitor acts as a competitive inhibitor for NTHi Nuc when low concentration of the substrate was present, as measured by FRET. Bars represent the average from three independent experiments for 0.03nM of pdTp and two independent experiments for 0.015nM of pdTp ± SD. p-values were determined using two-sided student’s t test with assumption of equal variance (*** p-value < 0.0005).

Fig 5. Effect of Co²⁺ on NTHi Nuc structure and activity.

NTHi Nuc with Ca²⁺ has a hydrodynamic radius of approximately 2.1 nm when in solution. Within minutes of adding Co²⁺, a known inhibitor for staphylococcal thermonuclease, the size of NTHi Nuc increased dramatically indicating oligomerization or nonspecific aggregation (solid line in Panel A). The aggregates formed by the addition of Co²⁺ to NTHi Nuc were briefly disrupted by passing the solution through a 0.22 μm membrane filter reduced the hydrodynamic radius to approximately 15 nm (start of dashed line in Panel A). However, NTHi Nuc continued aggregating and increasing in size due to Co²⁺ still present in solution (dashed line). Panel A shows representative curves. Activity of NTHi Nuc was quenched with addition of Co²⁺ when measured by FRET, similar to when EDTA was added (Panel B). Panel A shows representative curves and Panel B was done in triplicates.

Fig 6. SAXS analysis of NTHi Nuc.

Fig 6A shows scaled SAXS scattering curves of NTHi Nuc in pH = 7 (blue) and 9 (red). There were no gross structural changes at those two pH. Fig 6B and Fig 6C show Guinier plot for NTHi Nuc at pH = 7 and 9, respectively. Rg was 17.4 Å at both pH. Fig 6D shows parabolic Kratky plots in pH 7 (blue) and 9 (red), showing some flexible region. Fig 6E shows pairwise distribution function plots at pH 7 (blue) and 9 (red), with maximum dimensions of 58.3 Å at pH 7 and 59.6 Å at pH 9.

Fig 7. Structure of NTHi Nuc by SAXS.

Fig 7A shows a homology model of NTHi Nuc shown as a cartoon, blue = N-terminal and red = C-terminal. Fig 7B shows the ab initio shape of NTHi Nuc determined from SAXS data at pH 7. Fig 7C shows the surface representation of staphylococcal thermonuclease crystal structure. Fig 7B and 7C show similar overall shape and active site (black arrow).

Fig 8. Homology model comparison of NTHi Nuc to staphylococcal thermonuclease.

Overlay of the homology model of NTHi Nuc (blue), and the active site of staphylococcal thermonuclease (pdb id 1STN) (red). Green sphere represents calcium and cyan sticks represent TdtP (inhibitor); modeled from pdb id 2SNS. There are three loops in the active site of NTHi Nuc that are shorter than those of staphylococcal thermonculease. The absence of Ω loop near the active site of NTHi Nuc compared to the staphylococcal thermonuclease could contribute to the stronger activity of NTHi Nuc.