EmrE dimerization depends on membrane environment

AbstractThe 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 (KMD2D) of 0.002–0.009mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the KMD2D is only 0.8–0.95mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes KMD2D is 0.0005–0.0008mol% 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

Determination of Α-helix and Β-sheet stability in the solid state: A solid-state NMR investigation of poly( L -alanine)

The relative stability of Α-helix and Β-sheet secondary structure in the solid state was investigated using poly( L -alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. 13 C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the Α-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% Β-sheet and 45% Α-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% Β-sheet and 40% Α-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% Α-helix/50% Β-sheet at 100°C when starting from a mostly Α-helical state. No change was observed upon heating a Β-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with Β-sheet approximately 260 J/mol more stable than Α-helix in solid-state PLA. © 2002 Wiley Periodicals, Inc. Biopolymers 64: 246–254, 2002Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34326/1/10180_ftp.pd

Identification of an alternating-access dynamics mutant of EmrE with impaired transport

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue to Val, Ala, or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64V mutant identified here

Red blood cell invasion by Plasmodium vivax: Structural basis for DBP engagement of DARC

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction

A study of a C Α, Β -didehydroalanine homo-oligopeptide series in the solid-state by 13 C cross-polarization magic angle spinning NMR

The fully extended peptide conformation (2.0 5 -helix) has been investigated for the first time in the solid-state by 13 C cross-polarization magic angle spinning NMR. The compounds examined are members of a terminally protected, homo-oligopeptide series (from monomer through hexamer) based on C Α, Β -didehydroalanine. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35198/1/551_ftp.pd

Asymmetric protonation of EmrE

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with (1)H-(15)N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

The mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37.

Solid-state NMR and differential scanning calorimetry were used to investigate the mechanism of lipid bilayer disruption induced by LL-37, an amphipathic alpha-helical, antimicrobial peptide found in humans. The secondary structure, orientation, and dynamics of LL-37 in lipid bilayers were determined using solid-state NMR of site-specifically 13C, 15N, or 2H labeled LL-37. The effect of LL-37 on the lipid molecules was studied with 31P NMR of the lipid headgroup, 2H NMR of the acyl chains, and DSC. The results show that the amphipathic helical region of LL-37 is parallel to the bilayer surface and embedded in the hydrophobic/hydrophilic interface, interacting with both the lipid headgroups and acyl chains. The N-terminal nonhelical region is also associated with the membrane. LL-37 induces positive curvature strain in lipid bilayers, and alters their material properties, such as hydrophobic thickness and area per lipid. In DMPC, LL-37 increases the magnitude of the coefficient of thermal expansion both parallel and perpendicular to the bilayer normal. The orientation of LL-37 is the same regardless of the membrane environment, but the type and extent of headgroup and acyl chain perturbation depends on the lipid charge, headgroup type, acyl chain saturation, presence of cholesterol, and the presence of aqueous ions. This indicates that aggregation of LL-37 into regions of high local concentration and depth of insertion in the membrane interface vary with the relative balance of electrostatic and hydrophobic interactions between the peptide and the lipid headgroups and acyl chains, respectively. Of the mechanisms proposed for bilayer disruption by amphipathic a-helical peptides, the surface orientation and lack of small membrane fragments rule out the barrel-stave and detergent-like micellization mechanisms. All of the results support a toroidal pore model for LL-37 activity in lipid membranes.Ph.D.BiochemistryBiological SciencesBiophysicsPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123977/2/3106185.pd