Rotamer libraries of spin labelled cysteines for protein studies

Polyhach, Yevhen; Bordignon, Enrica; Jeschke, Gunnar

doi:10.1039/c0cp01865a

Cited by 411 publications

(592 citation statements)

References 51 publications

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“…Distance distributions were simulated using a rotamer library approach (RLA) as implemented in MMM 15 . The rotamer library consists of 210 precalculated rotamers representing an ensemble of possible spinlabel side chain (R1) conformations at 175 K. The orientation of R1 introduced at the chosen residue with regard to the protein structure permits calculation of the energy for the R1-protein interaction in consideration of the LennardJones potential 26 .…”

Section: Methodsmentioning

confidence: 99%

“…We constructed two trimerization-domain mutants by replacing Thr166 (located in the cytoplasmic end of transmembrane helix 4) and Val176 (located in the cytoplasmic end of helix 5) with an MTSL-modified cysteine (R1). Simulations on the available crystal structures indicated that these spin labels are >1.8 nm apart and therefore suitable for DEER measurements 15 . We did not observe spectral broadening in the CW EPR measurements, which further supports the notion that the spin labels were >1.8 nm apart (Supplementary Fig.…”

Section: Design Of Epr Measurementsmentioning

confidence: 99%

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Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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104

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Glt Ph is a Pyrococcus horikoshii homotrimeric Na + -coupled aspartate transporter that belongs to the glutamate transporter family. Each protomer consists of a trimerization domain involved in subunit interaction and a transporting domain with the substrate-binding site. Here, we have studied the conformational changes underlying transport by Glt Ph using EPR spectroscopy. The trimerization domains form a rigid scaffold, whereas the transporting domains sample multiple conformations, consistent with large-scale movements during the transport cycle. Binding of substrates changed the occupancies of the different conformational states, but the domains remained heterogeneous. The membrane environment favored conformations different from those observed in detergent micelles, but the transporting domain remained structurally heterogeneous in both environments. We conclude that the transporting domains sample multiple conformational states with substantial occupancy regardless of the presence of substrate and coupling ions, consistent with equilibrium constants close to unity between the observed transporter conformations. npg © 2013 Nature America, Inc. All rights reserved.nature structural & molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 2 1 1 a r t i c l e s of 160 K to qualitatively extract information on interspin distances <1.8 nm (ref. 14). We analyzed Glt Ph both solubilized in detergent micelles and reconstituted in proteoliposomes and compared the results from each experiment type. The trimerization domain is a stable scaffoldIntroduction of a spin label at a single position in Glt Ph allows for the measurement of the distance between the protomers of the homotrimeric protein. We constructed two trimerization-domain mutants by replacing Thr166 (located in the cytoplasmic end of transmembrane helix 4) and Val176 (located in the cytoplasmic end of helix 5) with an MTSL-modified cysteine (R1). Simulations on the available crystal structures indicated that these spin labels are >1.8 nm apart and therefore suitable for DEER measurements 15 . We did not observe spectral broadening in the CW EPR measurements, which further supports the notion that the spin labels were >1.8 nm apart (Supplementary Fig. 1). We recorded spectra of the apoprotein and of the protein in the presence of saturating concentrations of Na + alone or Na + and aspartate. In agreement with previous biochemical and structural data 12,13 , the trimerization domain did not show any large-scale conformational changes upon addition of coupling ions or substrate (Fig. 2). We observed two sharp peaks (centered around 2.8 nm and 3.8 nm for T166R1 and 2.6 nm and 3.4 nm for V176R1) in the interspin distance distributions for proteins in the presence and absence of substrates. The measured distances were in agreement with the interprotomer distances calculated on the basis of the crystal structures (Table 1 and Fig. 2).Because Glt Ph is a trimer, each mutation resulted in the presence of three cysteines per protein complex, which could result in a cons...

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Section: Methodsmentioning

confidence: 99%

Section: Design Of Epr Measurementsmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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104

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“…DEER traces were recorded at Q band with all pulses set to 12 ns and frequency separation of 100 MHz (37). Data analysis and simulation of the MTSL rotamers on the X-ray structures were performed with the softwares DeerAnalysis2013 (38) and MMM2013.2 (19). The D-loop aspartates of TM287/288 and LmrCD were substituted by alanines using site-directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

Höhl

Hürlimann

Böhm

et al. 2014

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

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ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.membrane transport | X-ray crystallography | allosteric communication A BC exporters are found in every organism (1, 2). They minimally consist of four domains and exist as homodimers or heterodimers. Two transmembrane domains (TMDs) span the membrane with a total of 12 transmembrane helices and form the substrate permeation pathway by alternating between inward-and outward-oriented states (Fig. S1A). A pair of nucleotide binding domains (NBDs) is connected to the TMDs via coupling helices and drive conformational cycling of the transporter by binding and hydrolysis of ATP, a process which is linked to NBD dimerization and dissociation (3).In their closed state, the NBDs sandwich two ATP molecules at the dimer interface by composite ATP binding sites involving conserved sequence motifs contributed by both subunits (4, 5). The A-loop and Walker A motif of one NBD and the ABC signature motif of the opposite NBD are involved in nucleotide binding. The Walker B glutamate and the switch-loop histidine constitute a catalytic dyad required for ATP hydrolysis (6, 7). In heterodimeric ABC exporters with asymmetric ATP binding sites, these catalytic residues are noncanonical at the degenerate site and ATP is therefore primarily, if not exclusively, hydrolyzed at the consensus site (8). The Q-and D...

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“…1 B; Table S2). We applied in silico modeling of the internal conformational flexibility of the MTSSL using rotamer libraries obtained from different programs (47,53) to simulate distance distributions and intramolecular dipolar evolution functions to compare our results to the available x-ray structure. Comparison of the experimental distance distributions with predicted distributions based on the x-ray structure (19), using MMM in 298 K mode, shows that experimental and predicted mean distances are in good agreement (Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…The spin label (SL) utilized, MTSSL ((1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate)), possesses an intrinsic linker flexibility, which needs to be taken into account when a structure is refined or modeled based on PELDOR distance constraints. Several approaches to describe the intrinsic flexibility of the SL, such as rotamer libraries, as well as molecular dynamics (MD) simulations, have been introduced to enhance the reliability of structural modeling (46)(47)(48). We used site-directed spin labeling (SDSL) to investigate the relative orientation and conformational flexibility between POTRA domains of anaOmp85 by PELDOR spectroscopy using 27 double cysteine mutants including intra-and interdomain distance constraints.…”

Section: Introductionmentioning

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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Rotamer libraries of spin labelled cysteines for protein studies

Cited by 411 publications

References 51 publications

Conformational heterogeneity of the aspartate transporter GltPh

Conformational heterogeneity of the aspartate transporter GltPh

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

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

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