Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

Abstract

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 b-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal b-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 POTRAdomain-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 spinlabeled 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.

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85

“…Further, the interaction with the N‐terminus of ana Omp85‐I is consistent with the notion that the POTRA domains are deeply embedded in the peptidoglycan layer (PGL). In the current model, the POTRA2 and POTRA3 are buried in the PGL, while POTRA1 and the N‐terminus face the periplasmatic surface of the PGL (Dastvan et al , ). Thus, ana Tic22 would be able to interact with the N‐terminus without entering the PGL.…”

“…This indicates that the three Omp85 proteins are not entirely functionally redundant. The three POTRA domains of ana Omp85‐I are arranged in an extended conformation (Koenig et al , ; Dastvan et al , ). Consistent to the postulated functions of the proteobacterial POTRA domains, the POTRAs of ana Omp85‐I were shown to bind to an artificial substrate (Ertel et al , ).…”

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains

“…Characterization of conformations and flexibilities of such multi-domain proteins requires the measurement of long-range distance restraints in the nanometer range, which are accessible by PELDOR spectroscopy. Since PELDOR time traces are sensitive to the conformational flexibility of both spin label and protein molecule (Dastvan et al, 2016;Jeschke, 2013;Klose et al, 2012;Polyhach et al, 2010), we used doubly spin-labeled monoubiquitin to assess the inherent intra-ubiquitin conformational flexibility (Figures S1A and S1B). Subsequently, we investigated how different linkage types of diubiquitin chains influence the conformational space by attaching one spin label to each ubiquitin moiety.…”

The contribution of modern EPR to structural biology

“…For more complex conformational transitions, one may resort to comparative modelling by satisfaction of spatial restraints as implemented in MODELLER [43]. Conformational changes in the multidrug transporter EmrE with respect to the crystal structure and upon protonation were assessed using the MMM interface to MODELLER that allows for imposing distance restraints between spin-labelled sites [44]. By the same approach, additional restraints on secondary structure can be included, as shown for the external loop eL4 in proline/sodium symporter PutP [45].…”