M. Gregor Madej

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (‘proton‐motive force’), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem‐containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non‐functionality of this novel transmembrane proton transfer pathway (‘E‐pathway’) in a variant QFR where a key glutamate residue has been replaced. The ‘E‐pathway’, discussed on the basis of the 1.78‐Å‐resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic ε‐proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem‐containing membrane proteins, performing very different physiological functions.

Functional architecture of MFS d-glucose transporters

Significance The crystallographic model of the Major Facilitator Superfamily (MFS) member, d-xylose permease XylE from Escherichia coli, a homologue of human d-glucose transporters, the GLUTs (SLC2), provides a structural framework for the identification and physical localization of crucial residues in transporters with medical relevance (i.e. the GLUTs). The mechanism and substrate specificity of human and prokaryotic sugar transporters are discussed by using homology modeling, molecular docking, and experimentation. Substrate-specificity determinants for XylE, GLUT1, and GLUT5 are proposed. Furthermore, concepts derived from other bacterial MFS transporters are examined for their relevance to the GLUTs by comparing conservation of critical residues. XylE mutants that mimic the characteristics of GLUT1 are tested, revealing that uniport and symport are mechanistically related. The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens. One objective of determining crystallographic models of the bacterial representatives is identification and physical localization of residues important for catalysis in transporters with medical relevance. The recently solved crystallographic models of the d-xylose permease XylE from Escherichia coli and GlcP from Staphylococcus epidermidus, homologs of the human d-glucose transporters, the GLUTs (SLC2), provide information about the structure of these transporters. The goal of this work is to examine general concepts derived from the bacterial XylE, GlcP, and other MFS transporters for their relevance to the GLUTs by comparing conservation of functionally critical residues. An energy landscape for symport and uniport is presented. Furthermore, the substrate selectivity of XylE is compared with GLUT1 and GLUT5, as well as a XylE mutant that transports d-glucose.

Apo-intermediate in the transport cycle of lactose permease (LacY)

The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H+. Crystal structures reveal 12, mostly irregular, transmembrane α-helices surrounding a cavity with sugar- and H+- binding sites at the apex, which is accessible from the cytoplasm and sealed on the periplasmic side (an inward-facing conformer). An outward-facing model has also been proposed based on biochemical and spectroscopic measurements, as well as the X-ray structure of a related symporter. Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism. Here, we generate a model for an apo-intermediate of LacY based on crystallographic coordinates of LacY and the oligopeptide/H+ symporter. The model exhibits a conformation with an occluded cavity inaccessible from either side of the membrane. Furthermore, kinetic considerations and double electron-electron resonance measurements suggest that another occluded conformer with bound sugar exists during turnover. An energy profile for symport is also presented.

Design, synthesis, and biological testing of novel naphthoquinones as substrate-based inhibitors of the quinol/fumarate reductase from Wolinella succinogenes.

Novel naphthoquinones were designed, synthesized, and tested as substrate-based inhibitors against the membrane-embedded protein quinol/fumarate reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure-activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b-k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a-c) and 2-methoxy-1,4-naphthoquinone (5a-b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50 values in low micromolar range in the primary assay and no activity in the counter DT-diaphorase assay.

Substrate-induced changes in the structural properties of LacY

Significance Lactose permease of Escherichia coli (LacY), a model for the major facilitator superfamily, catalyzes galactopyranoside/H+ symport across the membrane by a mechanism in which large conformational changes expose the sugar-binding site in the middle of the molecule alternatively to either side of the membrane (an alternating access mechanism). Despite substantial progress with respect to static X-ray crystal structures of LacY, the dynamics of the transport mechanism are not fully understood. Here we use dynamic single-molecule force spectroscopy to quantify the structural properties that change upon substrate binding. The results reveal very significant changes in conformational, kinetic, energetic, and mechanical properties primarily in the N-terminal 6-helix bundle, while the C-terminal 6-helix bundle remains largely unaffected. The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H+-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H+- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H+ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.

An Early Event in the Transport Mechanism of LacY Protein INTERACTION BETWEEN HELICES V AND I

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg144 and Trp151, two residues that play direct roles in sugar recognition and binding, as well as Cys154, which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro28 → Cys/Cys154, Pro28 → Cys/Val158 → Cys, and Phe29 → Cys/Val158 → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)2-1), 3–4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)2-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.

The correlation of cathodic peak potentials of vitamin K3 derivatives and their calculated electron affinities: The role of hydrogen bonding and conformational changes

2-methyl-1,4-naphtoquinone 1 (vitamin K(3), menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E(1/2)) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E(A)). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E(A)) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.

2-Hydroxy-3-(3-oxobutyl)naphthalene-1,4-dione.

The title compound, C14H12O4, forms crystals which appear monoclinic but are actually twinned triclinic. The asymmetric unit consists of two similar molecules, which differ only in the conformation of the 3-oxobutyl side chain. The molecular conformation is characterized by an intramolecular O-H...O hydrogen bond between the hydroxy group and the adjacent carbonyl O atom. The crystal structure is stabilized by O-H...O hydrogen bonds connecting the molecules into zigzag chains running along the b axis.