Characterization of the N-Terminal Domain of BteA: A <em>Bordetella</em> Type III Secreted Cytotoxic Effector

<div><p>BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical <em>Bordetella</em>, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s) and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA) in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287) and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR) experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.</p> </div

Secondary structure prediction of BteA superposed on established experimental data.

<p>The sequence of BteA was subjected to secondary structure meta-server analysis which was plotted as line for coil, spring for alpha-helix and arrow for beta sheet. Vertical arrows mark the boundaries of BteA287 fragment. Experimentally-derived secondary structure topology (PDB code 2JPF, aa 121–220) is shown in red. BteA-derived peptides from peptide fingerprinting MS/MS experiment (see Materials and Methods) were plotted as blue lines.</p

Size and oligomeric form of BteA287.

<p>(<b>A</b>) Size exclusion chromatogram of BteA287 demonstrates a single population. The elution volume of BteA287 combined with the calibration curve (inset) indicates an empirical molecular weight of 68 kDa (black point indicates the elution volume of BteA287 along the curve). (<b>B</b>, <b>C</b>) Bottom, sedimentation equilibrium curve for a 1 mg·ml⁻¹ (30 µM) BteA287 sample at 15,000 rpm and 298 K in buffer containing 100 mM NaCl, 20 mM Tris pH 8. The curve was fit to a model assuming either a single monomeric species or monomer-dimer equilibrium, in both cases in the presence of a high-MW aggregate. Top, residuals of the fit. Calculated mass was 34,500 Da. (<b>D</b>) LED-BPP-based experiments were acquired to estimate the translational diffusion coefficient of BteA at 298 K. The decrease in intensity of methyl region signals is plotted against (γGτ)², where γ is the proton gyromagnetic ratio (2.67·10⁸ T⁻¹s⁻¹), G is the applied magnetic gradient field and τ is the total length of the bipolar gradient, 4.8 ms in these experiments. Using the literature value of Ds = 11.1·10⁻¹¹ m²s⁻¹ for lysozyme (black) the value of G at maximal strength was determined as 0.514 Tm⁻¹. Curves shown are for 250 µM BteA287 (solid blue), 80 µM BteA287 (dotted blue), 250 µM BteA32-287 (solid red), and 80 µM BteA32-287 (dotted red). In the case of BteA287, mono-exponential fits can be significantly improved using a bi-exponential fit, indicating the presence of aggregation for the longer protein.</p

Summary of biophysical findings for BteA287.

1<p>Values in parentheses relates to high protein concentration (>200 µM).</p>2<p>Radius of hydration.</p>¥<p>Radius of gyration of 1 mg/ml sample.</p>¶<p>Radius of gyration of 2 mg/ml sample.</p

SAXS analysis of the BteA287.

<p>(<b>A</b>) Experimental data of the BteA287 (dots represent experimental data, and lines show fit) (<b>B</b>) Dummy-ball models (DBMs) of mg/ml (30 µM, upper panel) and 2 mg/ml (60 µM, lower panel) fitted onto the core deposited structure of BteA_115–220 (PDB code 2JPF).</p

Expression and purification analysis of BteA287.

<p>(<b>A</b>) SDS-PAGE analysis of non-induced (NI) and IPTG-induced (I) samples that were taken from overnight grown cultures and were lysed by the addition of sample buffer followed by SDS-PAGE. (<b>B</b>) SDS-PAGE analysis of BteA287 Ni²⁺-affinity chromatography (AC) and anionic ion-exchange chromatography (IEC) purifications. Lanes are labeled as follows, M, marker; WC, whole cell lysate; P, pellet; FT, flow through; Ni, Elution from AC column; Mq, elution from IEC column; Fi, concentrated BteA287 sample. (<b>C</b>) MALDI/TOF-MS analysis of purified BteA287 with corresponding masses (in Daltons) indicated above main peaks.</p

Circular dichroism analysis of BteA287 and 2D ¹H,¹⁵N-TROSY-HSQC spectrum of BteA287.

<p>(<b>A</b>) Dichroic spectra for BteA287 (black line) and BteA32-287 (blue line). <b>(B)</b> Melting curve of BteA287, measured at 222 nm (blue line indicates calculated Tm). (<b>C</b>) Spectrum was acquired for a triply-labeled [²H,¹³C,¹⁵N]-BteA287 0.4 mM sample in 100 mM NaCl, 20 mM NaPi pH 7.3 at 303 K and 16.4 T. The inset shows an outlying indole NH peak.</p

SDS-PAGE analysis of BteA purification and limited proteolysis experiments.

<p>(<b>A</b>) Ni²⁺-affinity chromatography purification of BteA showing fractions of: 1, pellet; 2, flow through; 3, wash 1; 4, wash 2; 5, wash 3; Elutions, selected elution fractions; M, marker. Black and white arrows indicate BteA monomer and oligomer respectively. (<b>B</b>) Limited proteolysis experiment was conducted on purified BteA after which the reaction was quenched by the addition of sample buffer and separated on 12.5% SDS-polyacrylamide.</p