BCL::MP‐fold: Membrane protein structure prediction guided by <scp>EPR</scp> restraints

Fischer, Anastasia; Woetzel, Nils; Karakaş, Mert; Weiner, Brian E.; Meiler, Jens

doi:10.1002/prot.24801

Cited by 25 publications

(49 citation statements)

References 53 publications

(136 reference statements)

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“…A twofold symmetry was imposed such that both protomers have similar conformations, in contrast to the crystal structure, where the two protomers have distinct conformations and form an asymmetric dimer. However, despite the extensive nature of the distance restraints and their coverage along the protein sequence, compatibility of the DEER data with an asymmetric dimer cannot be excluded-primarily due to the uncertainty in translation of measured distances between the spin labels into backbone structural restraints (23,25). However, as discussed below, we consider the asymmetric dimer to be less mechanistically plausible for the protonated state.…”

Section: Discussionmentioning

confidence: 97%

“…To highlight the conformational changes induced by protonation in a structural context, we carried out detailed de novo modeling of the pH 5 conformation using BCL::Fold and Rosetta (22)(23)(24)(25). The conformational search was restrained by the experimental distances at pH 5 (22,23).…”

Section: Discussionmentioning

confidence: 99%

“…The conformational search was restrained by the experimental distances at pH 5 (22,23). A twofold symmetry was imposed such that both protomers have similar conformations, in contrast to the crystal structure, where the two protomers have distinct conformations and form an asymmetric dimer.…”

Section: Discussionmentioning

confidence: 99%

“…The initial model of the EmrE structure at pH 5 was predicted by a two-step approach using BCL::Fold (22,23) to assemble the secondary structure elements (SSEs) in 3D space and Rosetta (24,25) to construct loop regions and predict sidechain conformations. See SI Methods for more details on BCL/Rosetta modeling.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

Dastvan

Fischer

Mishra

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the protonbound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward-and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.EmrE | SMR | EPR | DEER | multidrug transport P owered by the proton electrochemical gradient across the inner membrane of prokaryotes, small multidrug resistance (SMR) transporters extrude a spectrum of cytotoxic molecules that are primarily hydrophobic cations (1). The functional unit is typically a homodimer wherein each protomer consists of four hydrophobic transmembrane helices (TM). TM1-3 cradle a substrate binding pocket, whereas TM4 is involved in the contacts that stabilize the dimer. EmrE, the SMR transporter from Escherichia coli, has been a focal point of structural, spectroscopic, and mechanistic investigations (2-7). Seminal work from the Schuldiner laboratory over the last two decades has unlocked mechanistic principles of substrate-ionantiport (1,(8)(9)(10). EmrE binds hydrophobic substrates in a membrane-embedded chamber coordinated by glutamate 14 (E14), an absolutely conserved, membrane-embedded, acidic residue in the middle of TM1. Coupling between substrate and proton arises from the principle of mutual exclusion between the two ligands at the binding site (8,10).In contrast to the elaborate understanding of the EmrE antiport mechanism, the conformational changes that enable binding and release of ligands have not been elucidated. Although the general framework of antiport is presumed to follow the principles of alternating access, the only structure available is of EmrE bound to the substrate tetraphenylphosphonium (TPP) (3). EM analysis of 2D crystals established an antiparallel ...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

Dastvan

Fischer

Mishra

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…S16). Furthermore, due to the inherent flexibility of the SL, and thus the uncertainty in prediction of the mean label position with respect to the protein backbone, structural refinement cannot be improved higher than a limit (70).…”

Section: Discussionmentioning

confidence: 99%

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

Dastvan

Brouwer

Schuetz

et al. 2016

Biophysical Journal

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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.

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CASP10-BCL::Fold efficiently samples topologies of large proteins

et al. 2015

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During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold’s ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein.

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BCL::MP‐fold: Membrane protein structure prediction guided by EPR restraints

Cited by 25 publications

References 53 publications

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

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

CASP10-BCL::Fold efficiently samples topologies of large proteins

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