Crystal Structures and Mutational Analysis of the Arginine-, Lysine-, Histidine-binding Protein ArtJ from Geobacillus stearothermophilus. Implications for Interactions of ArtJ with its Cognate ATP-binding Cassette Transporter, Art(MP)2
ArtJ is the substrate-binding component (receptor) of the ATP-binding cassette (ABC) transport system ArtJ-(MP)(2) from the thermophilic bacterium Geobacillus stearothermophilus that is specific for arginine, lysine, and histidine. The highest affinity is found for arginine (K(d)=0.039(+/-0.014) microM), while the affinities for lysine and histidine are about tenfold lower. We have determined the X-ray structures of ArtJ liganded with each of these substrates at resolutions of 1.79 A (arginine), 1.79 A (lysine), and 2.35 A (histidine), respectively. As found for other solute receptors, the polypeptide chain is folded into two distinct domains (lobes) connected by a hinge. The interface between the lobes forms the substrate-binding pocket whose geometry is well preserved in all three ArtJ/amino acid complexes. Structure-derived mutational analyses indicated the crucial role of a region in the carboxy-terminal lobe of ArtJ in contacting the transport pore Art(MP)(2) and revealed the functional importance of Gln132 and Trp68. While variant Gln132Leu exhibited lower binding affinity for arginine but no binding of lysine and histidine, the variant Trp68Leu had lost binding activity for all three substrates. The results are discussed in comparison with known structures of homologous proteins from mesophilic bacteria.
"Based on analogy with ArtJ, there are two main regions comprised of residues 42–51 in lobe I and residues 171–177 in lobe II that play a major role in this process. Of particular interest is the observation that Glu163 of ArtJ, which was found by mutagenesis analyses to be a key player in this recognition process , is conserved in TmArgBP (Glu171). "
[Show abstract][Hide abstract] ABSTRACT: The arginine binding protein from Thermatoga maritima (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.
PLoS ONE 05/2014; 9(5):e96560. DOI:10.1371/journal.pone.0096560 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The thermostable arginine ABC transporter of Geobacillus stearothermophilus consists of a solute binding protein, ArtJ; a transmembrane subunit, ArtM; and a nucleotide-binding subunit, ArtP. An ArtM/His(6)-ArtP complex was functionally assembled from separately purified subunits as demonstrated by assaying stimulation of its ATPase activity by arginine-loaded ArtJ in proteoliposomes. Studying in vitro assembly with variants carrying mutations in the conserved Q loop and/or the EAA loop of ArtP and ArtM, respectively, confirmed the predicted roles of both motifs in intersubunit signaling and physical interaction, respectively. In vitro assembly is considered a useful tool for investigating assembly defects of ABC transporters caused by mutations.
[Show abstract][Hide abstract] ABSTRACT: This paper identifies the first arginine/ornithine antiporter ArcD from the domain of archea. The functional role of ArcD is demonstrated by transport assays with radioactive labelled arginine, by its necessity to enable arginine fermentation under anaerobic growth conditions and by the consumption of arginine from the medium during growth. All three experimentally observables are severely disturbed when the deletion strain DeltaArcD is used. The isolated protein is verified by mass spectrometry and reconstituted in vesicles. The proteoliposomes are attached to a membrane and capacitive currents are recorded which appear upon initiation of the transport process by change from arginine-free to arginine-containing buffer. This clearly demonstrates that the purified 34kD protein is the functional unit.
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