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Institute
Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.
Relative orientation of POTRA domains from cyanobacterial Omp85 studied by pulsed EPR spectroscopy
(2016)
Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a β-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.
Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most informations on protein translocation are obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a primitive translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has subsequently been lost in the transit sequence, but can be found at the C-terminal position of the translocating pore. Thereby, the current hypothesis of Omp85 being the prokaryotic contribution to the ancestral Toc translocon can be supported.
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.