X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB.
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ABSTRACT:
SUBMITTER: Guan L
PROVIDER: S-EPMC8329300 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The bacterial melibiose permease (MelB) belongs to the glycoside-pentoside-hexuronide:cation symporter family, a part of the major facilitator superfamily (MFS). Structural information regarding glycoside-pentoside-hexuronide:cation symporter family transporters and other Na(+)-coupled permeases within MFS has been lacking, although a wealth of biochemical and biophysical data are available. Here we present the three-dimensional crystal structures of Salmonella typhimurium MelBSt in two conformations, representing an outward partially occluded and an outward inactive state of MelBSt. MelB adopts a typical MFS fold and contains a previously unidentified cation-binding motif. Three conserved acidic residues form a pyramidal-shaped cation-binding site for Na(+), Li(+) or H(+), which is in close proximity to the sugar-binding site. Both cosubstrate-binding sites are mainly contributed by the residues from the amino-terminal domain. These two structures and the functional data presented here provide mechanistic insights into Na(+)/melibiose symport. We also postulate a structural foundation for the conformational cycling necessary for transport catalysed by MFS permeases in general.
| S-EPMC4026327 | biostudies-literature
Project description:BackgroundMembrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein.ResultsLipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy.Conclusions(1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.
| S-EPMC6091025 | biostudies-literature
Project description:The melibiose permease of Salmonella typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+), and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na(+)-binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding of cosubstrates depended on the physical-chemical properties of the side chain. Compared with WT MelB(St), the Gly117 ? Ala mutant exhibited little difference in either cosubstrate binding or stimulation of melibiose transport by Na(+) or Li(+), but all other mutations reduced melibiose active transport and efflux, and decreased the apparent affinity for Na(+). The bulky Trp at position 117 caused the greatest inhibition of melibiose binding, and Gly117 ? Arg yielded less than a 4-fold decrease in the apparent affinity for melibiose at saturating Na(+) or Li(+) concentration. Remarkably, the mutant Gly117 ? Arg catalyzed melibiose exchange in the presence of Na(+) or Li(+), but did not catalyze melibiose translocation involving net flux of the coupling cation, indicating that sugar is released prior to release of the coupling cation. Taken together, the findings are consistent with the notion that Gly117 plays an important role in cation binding and translocation.
| S-EPMC3334291 | biostudies-literature
Project description:Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 from Arabidopsis thaliana (AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2'-deoxycytidine 5'-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.
| S-EPMC5617298 | biostudies-literature
Project description:The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na(+) binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.
| S-EPMC3009771 | biostudies-literature
Project description:The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.
| S-EPMC3057838 | biostudies-literature
Project description:Efficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source, such as glucose, represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression. In enteric bacteria, the key player of carbon catabolite repression is a component of the glucose-specific phosphotransferase system, enzyme IIA (EIIA(Glc)). It is known that unphosphorylated EIIA(Glc) binds to and inhibits a variety of transporters when glucose is available. However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any EIIA(Glc)-transporter complexes. Here we present the 3.9?Å crystal structure of Escherichia coli EIIA(Glc) in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIA(Glc) molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half-maximal inhibitory concentrations of the full-length EIIA(Glc) and an amino-terminal truncation mutant differ by 60-fold, consistent with the hypothesis that the amino-terminal region, disordered in the crystal structure, functions as a membrane anchor to increase the effective EIIA(Glc) concentration at the membrane. Together these data suggest a model of how the central regulatory protein EIIA(Glc) allosterically inhibits maltose uptake in E. coli.
| S-EPMC3875231 | biostudies-literature
Project description:Numerous studies have shown how periplasmic binding proteins (PBPs) bind substrates with exquisite specificity, even distinguishing between sugar epimers and anomers, or structurally similar ions. Yet, marked substrate promiscuity is also a feature encoded in some PBPs. Except for three sub-Ångström crystal structures, there are no reports of hydrogen atom positions in the remaining (> 1000) PBP structures. The previous X-ray crystal structure of the maltodextrin periplasmic-binding protein from Thermotoga maritima (tmMBP) complexed with oligosaccharide showed a large network of interconnected water molecules stretching from one end of the substrate binding pocket to the other. These water molecules are positioned to form multiple hydrogen bonds, as well as forming interactions between the protein and substrate. Here we present the neutron crystal structure of tmMBP to a resolution of 2.1 Å. This is the first neutron crystal structure from the PBP superfamily and here we unambiguously identify the nature and orientation of the hydrogen bonding and water-mediated interactions involved in stabilizing a tetrasaccharide in the binding site. More broadly, these results demonstrate the conserved intricate mechanisms that underlie substrate-specificity and affinity in PBPs.
| S-EPMC9587032 | biostudies-literature
Project description:Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7-190 d, most of which (2-180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.
| S-EPMC6235626 | biostudies-literature
Project description:We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.
| S-EPMC7540567 | biostudies-literature