The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[³H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.     

In vitro Unfolding and Refolding of the Small Multidrug Transporter EmrE

The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process. EmrE is part of a multidrug resistance protein family that is highly conserved amongst bacteria and is responsible for bacterial resistance to toxic substances. We find that the secondary structure of EmrE is very stable and only small amounts are denatured even in the presence of unusually high denaturant concentrations involving a combination of 10 M urea and 5% SDS. Substrate binding by EmrE is recovered after refolding this denatured protein into dodecylmaltoside detergent micelles or into lipid vesicles. The yield of refolded EmrE decreases with lipid bilayer compositional changes that increase the lateral chain pressure within the bilayer, whilst conversely, the apparent rate of folding seems to increase. These results add further weight to the hypothesis that an increased lateral chain pressure hinders protein insertion across the bilayer. Once the protein is inserted, however, the greater pressure on the transmembrane helices accelerates correct packing and final folding. This work augments the relatively small number of biophysical folding studies in vitro on helical membrane proteins.     

The NorM MATE Transporter from N.?gonorrhoeae: Insights into Drug and Ion Binding from Atomistic Molecular Dynamics Simulations

The multidrug and toxic compound extrusion transporters extrude a wide variety of substrates out of both mammalian and bacterial cells via the electrochemical gradient of protons and cations across the membrane. The substrates transported by these proteins include toxic metabolites and antimicrobial drugs. These proteins contribute to multidrug resistance in both mammalian and bacterial cells and are therefore extremely important from a biomedical perspective. Although specific residues of the protein are known to be responsible for the extrusion of solutes, mechanistic details and indeed structures of all the conformational states remain elusive. Here, we report the first, to our knowledge, simulation study of the recently resolved x-ray structure of the multidrug and toxic compound extrusion transporter, NorM from Neisseria gonorrhoeae (NorM_NG). Multiple, atomistic simulations of the unbound and bound forms of NorM in a phospholipid lipid bilayer allow us to identify the nature of the drug-protein/ion-protein interactions, and secondly determine how these interactions contribute to the conformational rearrangements of the protein. In particular, we identify the molecular rearrangements that occur to enable the Na⁺ ion to enter the cation-binding cavity even in the presence of a bound drug molecule. These include side chain flipping of a key residue, GLU-261 from pointing toward the central cavity to pointing toward the cation binding side when bound to a Na⁺ ion. Our simulations also provide support for cation binding in the drug-bound and apo states of NorM_NG.     

Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent

We have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer.     

A quasi-elastic laser light scattering study of tubulin and microtubule protein from bovine brain

Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D_{20, W} = 3.1 × 10^?7 cm² s^?1 and the value of D_{20, W} = 5.1 × 10^?7 cm² s^?1 calculated for tubulin dimer of M_rel 100,000. The 30 S ring has an observed diffusion coefficient of D_{20, W} = 0.49 × 10^?7 cm² s^?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.     

Mechanisms for Two-Step Proton Transfer Reactions in the Outward-Facing Form of MATE Transporter

Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, various drugs or antibiotics are extruded across the cell membrane by multidrug transporters. The multidrug and toxic compound extrusion (MATE) transporter is one of the five families of multidrug transporters. MATE from Pyrococcus furiosus uses H⁺ to transport a substrate from the cytoplasm to the outside of a cell. Crystal structures of MATE from P. furiosus provide essential information on the relevant H⁺-binding sites (D41 and D184). Hybrid quantum mechanical/molecular mechanical simulations and continuum electrostatic calculations on the crystal structures predict that D41 is protonated in one structure (Straight) and, both D41 and D184 protonated in another (Bent). All-atom molecular dynamics simulations suggest a dynamic equilibrium between the protonation states of the two aspartic acids and that the protonation state affects hydration in the substrate binding cavity and lipid intrusion in the cleft between the N- and C-lobes. This hypothesis is examined in more detail by quantum mechanical/molecular mechanical calculations on snapshots taken from the molecular dynamics trajectories. We find the possibility of two proton transfer (PT) reactions in Straight: the 1st PT takes place between side-chains D41 and D184 through a transient formation of low-barrier hydrogen bonds and the 2nd through another H⁺ from the headgroup of a lipid that intrudes into the cleft resulting in a doubly protonated (both D41 and D184) state. The 1st PT affects the local hydrogen bond network and hydration in the N-lobe cavity, which would impinge on the substrate-binding affinity. The 2nd PT would drive the conformational change from Straight to Bent. This model may be applicable to several prokaryotic H⁺-coupled MATE multidrug transporters with the relevant aspartic acids.     

The Mycobacterial MsDps2 Protein Is a Nucleoid-Forming DNA Binding Protein Regulated by Sigma Factors σA and σB

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors σ^H and σ^F. We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing σ^A or σ^B, under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.     

The NorM MATE Transporter from N. gonorrhoeae: Insights into Drug and Ion Binding from Atomistic Molecular Dynamics Simulations

The multidrug and toxic compound extrusion transporters extrude a wide variety of substrates out of both mammalian and bacterial cells via the electrochemical gradient of protons and cations across the membrane. The substrates transported by these proteins include toxic metabolites and antimicrobial drugs. These proteins contribute to multidrug resistance in both mammalian and bacterial cells and are therefore extremely important from a biomedical perspective. Although specific residues of the protein are known to be responsible for the extrusion of solutes, mechanistic details and indeed structures of all the conformational states remain elusive. Here, we report the first, to our knowledge, simulation study of the recently resolved x-ray structure of the multidrug and toxic compound extrusion transporter, NorM from Neisseria gonorrhoeae (NorM_NG). Multiple, atomistic simulations of the unbound and bound forms of NorM in a phospholipid lipid bilayer allow us to identify the nature of the drug-protein/ion-protein interactions, and secondly determine how these interactions contribute to the conformational rearrangements of the protein. In particular, we identify the molecular rearrangements that occur to enable the Na⁺ ion to enter the cation-binding cavity even in the presence of a bound drug molecule. These include side chain flipping of a key residue, GLU-261 from pointing toward the central cavity to pointing toward the cation binding side when bound to a Na⁺ ion. Our simulations also provide support for cation binding in the drug-bound and apo states of NorM_NG.     

1H, 13C, 15N backbone NMR assignments of the Staphylococcus aureus small multidrug-resistance pump (Smr) in a functionally active conformation

The plasmid-encoded small multidrug resistance pump from S. aureus transports a variety of quaternary ammonium and other hydrophobic compounds, enhancing the bacterial host’s resistance to common hospital disinfectants. The protein folds as a homo-dimer of four transmembrane helices each, and appears to be fully functional only in lipid bilayers. Here we report the backbone resonance assignments and implied secondary structure for ²H¹³C¹⁵N Smr reconstituted into lipid bicelles. Significant changes were observed between the chemical shifts of the protein in lipid bicelles compared to those in detergent micelles.     

Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments

Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca²⁺ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca²⁺ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The ¹⁵N backbone and ¹³C_δ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo μs-ms dynamics (k_ex = 6100 ±800 s^- 1 at 17 °C). We measured k_ex at additional temperatures, which allowed for a calculation of activation energy equal to ∼ 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.     

EmrE dimerization depends on membrane environment

The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer–dimer equilibrium constant (K_MD^2D) of 0.002–0.009 mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the K_MD^2D is only 0.8–0.95 mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes K_MD^2D is 0.0005–0.0008 mol% based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity.     

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.     

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a ¹³C-labeled retinal cofactor and extensively ¹³C-¹⁵N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.     

Oligomeric-State-Dependent Conformational Change of the BLUF Protein TePixD (Tll0078)

The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 μs, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 × 10^− 11 to 4.4 × 10^− 11 m² s^− 1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10^− 11 m² s^− 1 upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.     

Cellular prion protein and Alzheimer disease

Soluble oligomeric amyloid-β (Aβ) has been suggested to impair synaptic and neuronal function, leading to neurodegeneration that is clinically observed as the memory and cognitive dysfunction characteristic of Alzheimer disease, while the precise mechanism(s) whereby oligomeric Aβ causes neurotoxicity remains unknown. Recently, the cellular prion protein (PrP^C) was reported to be an essential co-factor in mediating the neurotoxic effect of oligomeric Aβ. Our recent study showed that Prnp^−/− mice are resistant to the neurotoxic effect of oligomeric Aβ in vivo and in vitro. Furthermore, application of an anti-PrP^C antibody or PrP^C peptide was able to block oligomeric Aβ-induced neurotoxicity. These findings demonstrate that PrP^C may be involved in neuropathologic conditions other than conventional prion diseases, i.e., Creutzfeldt-Jakob disease.     

Structure of a sigma28-regulated nonflagellar virulence protein from Campylobacter jejuni

Campylobacter jejuni, a Gram-negative motile bacterium, is a leading cause of human gastrointestinal infections. Although the mechanism of C.jejuni-mediated enteritis appears to be multifactorial, flagella play complex roles in the virulence of this human pathogen. Cj0977 is a recently identified virulence factor in C. jejuni and is expressed by a σ²⁸ promoter that controls late genes in the flagellar regulon. A Cj0977 mutant strain is fully motile but significantly reduced in the invasion of intestinal epithelial cells in vitro. Here, we report the crystal structure of the major structural domain of Cj0977, which reveals a homodimeric “hot-dog” fold architecture. Of note, the characteristic hot-dog fold has been found in various coenzyme A (CoA) compound binding proteins with numerous oligomeric states. Structural comparison with other known hot-dog fold proteins locates a putative binding site for an acyl-CoA compound in the Cj0977 protein. Structure-based site-directed mutagenesis followed by invasion assays indicates that key residues in the putative binding site are indeed essential for the Cj0977 virulence function, suggesting a possible function of Cj0977 as an acyl-CoA binding regulatory protein.     

Blocking Dynamics of the SMR Transporter EmrE Impairs Efflux Activity

EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.     

Blocking Dynamics of the SMR Transporter EmrE Impairs Efflux Activity

EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.