The Structure of HasB Reveals a New Class of TonB Protein Fold

<div><p>TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system. Despite a considerable amount of data available, the molecular mechanism of TonB-dependent active transport is still poorly understood. In this work, we present a structural study of a TonB-like protein, HasB dedicated to the HasR receptor. HasR acquires heme either free or <i>via</i> an extracellular heme transporter, the hemophore HasA. Heme is used as an iron source by bacteria. We have solved the structure of the HasB periplasmic domain of <i>Serratia marcescens</i> and describe its interaction with a critical region of HasR. Some important differences are observed between HasB and TonB structures. The HasB fold reveals a new structural class of TonB-like proteins. Furthermore, we have identified the structural features that explain the functional specificity of HasB. These results give a new insight into the molecular mechanism of nutrient active transport through the bacterial outer membrane and present the first detailed structural study of a specific TonB-like protein and its interaction with the receptor.</p> </div

Analysis of HasB_CTD upon HasR TonB box peptide binding.

<p>A: ¹⁵N HSQC spectra of HasB_CTD in the presence (blue) or absence (black) of the TonB box peptide. B: CSP values shown by residue; C: CSP mapped onto HasB_CTD structure. Residues showing the greatest backbone amide shifts (>0.2 ppm) are shown in orange. Residues not seen in the ¹⁵N HSQC spectrum, due to intermediate chemical exchange, are indicated by negative bars in B and colored in red in C.</p

Structural comparison of HasB_CTD with TolA_CTD and TonB_CTD. A:

<p>Ribbon representation of available solution structure of TonB and TolA proteins family. <b>B:</b> ClustalW alignments of HasB_CTD and TonB_CTD with their corresponding secondary structure. Cylinders represent the helices and arrows the β-strands of HasB_CTD structure. The secondary structure elements of other proteins are from a consensus prediction generated by <a href="mailto:NPS@" target="_blank">NPS@</a> (Combet C, Blanchet C, Geourjon C, & Deléage, G (2000). <a href="mailto:NPS@" target="_blank">NPS@</a>: network protein sequence analysis. <i>Trends. Biochem. Sci. </i><b>25</b>, 147–150). Predicted helices and β-strands are respectively presented in black on gray background and in white on black background. (*) conserved residues; (:) similar residues. The sequence numbering is that of HasB_CTD. H.i.: residues 143–264 of <i>Haemophilus influenzae</i> TonB (#68248858); P.m.: residues 133–256 of <i>Pasteurella multocida</i> TonB (#33318341); Y.e.: residues 133–256 of <i>Yersinia enterocolitica</i> TonB (#A1JIK8); H.p.: residues 155–279 of <i>Helicobacter pylori</i> TonB1341 (#889856).</p

TonB box interaction with HasB_CTD and TonB_CTD.

<p>The model of the complex HasB_CTD-TonB box (A) is compared to the structure of the complex TonB_CTD-TonB box (B). The TonB box of HasR is in orange, the BtuB TonB box (PDB code: 2GSK) is in yellow. Intermolecular ionic interactions are shown in spheres. The residues of HasB_CTD and TonB_CTD involved in the intermolecular ionic interaction with TonB box are indicated in bold (C).</p

Backbone dynamics of HasB_CTD that identifies regions of well-defined structure and mobility.

<p>Steady-state ¹⁵N-¹H NOE (A), relaxation rates R1 (B) and R2 (C) were measured at 500 MHz and 293 K.</p

ITC analysis of the interaction of HasB_CTD with the 21-mer peptide corresponding to HasR TonB box.

<p>A representative experiment is shown. The heat signal (top) is presented together with the binding isotherm derived from the signal (bottom). The concentration of HasB_CTD and the peptide were respectively, 9×10⁻⁵ M and 1.5 mM.</p

Summary of the NMR constraints used for the structure calculation, the restraint violations and structural statistics for the ensemble of 20 best conformers of HasB_CTD.

<p>Summary of the NMR constraints used for the structure calculation, the restraint violations and structural statistics for the ensemble of 20 best conformers of HasB_CTD.</p

Overall structure of HasB_CTD.

<p>A: Secondary structure elements in HasB_CTD. Arrows represent the β-strands and cylinders the α-helices. B: Ribbon diagram of backbone superposition of the 20 final structures of HasB_CTD. The N-terminal tail (residues 1–35) is not presented.</p

Structure and backbone dynamics of the periplasmic domain of HasR.

<p>(A) Overall structure of the HasR periplasmic domain. Top: cartoon diagram of the family of ten structures with lowest energy values (the flexible N- and C-termini are not presented). Bottom: (A) representative structure of the family (all residues are shown) with the linker containing the HasB/TonB box (magenta). The structure is rotated 180° around the Y axis. (B) Steady-state ¹⁵N-¹H NOE (C) relaxation rates R2 and (D) R1 were measured at 600 MHz at 20°C. (E) Structural comparison of the signaling domain of HasR with its two structural homologous. The loop 35–47 is surrounded.</p

Backbone dynamics of HasS_CTD.

<p>Steady state ¹⁵N-¹H (A) longitudinal and transverse relaxation rates R1 (B) and R2 (C) were measured at 600 MHz and 20°C. R2/R1 ratios were calculated from B and C.</p