Swapping dimer of HoloTmArgBP.

<p>(A) Cartoon representation of HoloTmArgBP domain-swapped dimer. (B) Omit (Fo-Fc) map of the hinge region contoured at 2σ.</p

A Loose Domain Swapping Organization Confers a Remarkable Stability to the Dimeric Structure of the Arginine Binding Protein from <i>Thermotoga maritima</i>

<div><p>The arginine binding protein from <i>Thermatoga maritima</i> (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate the atomic level structural properties of this intriguing protein, we determined the crystallographic structures of the apo and the arginine-bound forms of TmArgBP using MAD and SAD methods, respectively. The comparison of the liganded and unliganded models demonstrates that TmArgBP tertiary structure undergoes a very large structural re-organization upon arginine binding. This transition follows the Venus Fly-trap mechanism, although the entity of the re-organization observed in TmArgBP is larger than that observed in homologous proteins. Intriguingly, TmArgBP dimerizes through the swapping of the C-terminal helix. This dimer is stabilized exclusively by the interactions established by the swapping helix. Therefore, the TmArgBP dimer combines a high level of stability and conformational freedom. The structure of the TmArgBP dimer represents an uncommon example of large tertiary structure variations amplified at quaternary structure level by domain swapping. Although the biological relevance of the dimer needs further assessments, molecular modelling suggests that the two TmArgBP subunits may simultaneously interact with two distinct ABC transporters. Moreover, the present protein structures provide some clues about the determinants of the extraordinary stability of the biomolecule. The availability of an accurate 3D model represents a powerful tool for the design of new TmArgBP suited for biotechnological applications.</p></div

Domain-swapped dimer of ApoTmArgBP.

<p>(A) Cartoon representation of ApoTmArgBP swapping dimer. (B) Omit (Fo-Fc) map of the hinge region, contoured at 2σ. (C) Interactions mediated by the C-terminal helix. (D) Superposition of the chains A and B of ApoTmArgBP.</p

(2Fo-Fc) electron density map contoured around the arginine ligand (2.0 σ). Arginine interacting residues are highlighted.

<p>(2Fo-Fc) electron density map contoured around the arginine ligand (2.0 σ). Arginine interacting residues are highlighted.</p

Variation of tertiary (A) and quaternary (B) structures of ApoTmArgBP (orange) and HoloTmArgBP (magenta).

<p>In both panels, overlapped regions are 22–104 and 206–243 of lobe I. The arrows highlight the conformational changes occurring upon arginine binding.</p

Stability of the TmArgBP dimer.

<p>(A) Native PAGE electrophoresis of HoloTmArgBP and ApoTmArgBP. Lanes 1 and 2 contain HoloTmArgBP and ApoTmArgBP, respectively. The same experiments were carried out (Lanes 3 and 4) in the presence of 4M urea. (B) SDS PAGE upon treatment of Holo-TmArgBP and (C) Apo-TmArgBP with increasing urea concentrations. Lanes 1 and 2 contain urea concentrations 0 and 8 M, respectively. The same markers were used in the two experiments.</p

Isothermal titration calorimetry experiments with (A) arginine and (B) glutamine.

<p>Top panels report raw data for the titrations at 25°C, whereas bottom panels report integrated heats of binding obtained from the raw data after subtracting the heats of dilution. The solid line (in A) represents the best curve fit to the experimental data using the ‘one set of sites’ model from MicroCal Origin.</p

Analytical SEC-MALS of TmArgBP.

<p>The black and grey curves represent the Rayleigh ratio (left scale) of HoloTmArgBP and ApoTmArgBP, respectively; both are plotted against the retention time. Molecular masses are reported with the same colour code. In both experiments, average molecular masses values correspond to a dimeric state of the protein.</p

Structure and dynamics of the multi-domain resuscitation promoting factor RpfB from <i>Mycobacterium tuberculosis</i>

<p>RpfB is multidomain protein that is crucial for <i>Mycobacterium tuberculosis</i> resuscitation from dormancy. This protein cleaves cell wall peptidoglycan, an essential bacterial cell wall polymer formed by glycan chains of β-(1-4)-linked-N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by short peptide stems. RpfB is structurally complex being composed of five distinct domains, namely a catalytic, a G5 and three DUF348 domains. Here, we have undertaken a combined experimental and computation structural investigations on the entire protein to gain insights into its structure–function relationships. CD spectroscopy and light scattering experiments have provided insights into the protein fold stability and into its oligomeric state. Using the available structure information, we modeled the entire protein structure, which includes the two DUF348 domains whose structure is experimentally unknown, and we analyzed the dynamic behavior of RpfB using molecular dynamics simulations. Present results highlight an intricate mutual influence of the dynamics of the different protein domains. These data provide interesting clues on the functional role of non-catalytic domains of RpfB and on the mechanism of peptidoglycan degradation necessary to resuscitation of <i>M. tuberculosis</i>.</p

Multiple sequence alignment of TmArgBP with known homologues.

<p>For each protein the PDB code is reported in parenthesis. The signal peptide of TmArgBP is drawn in red. For the other proteins only the binding domain is reported.</p