Aromatic Stacking in the Sugar Binding Site of the Lactose Permease †

Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Biochemistry (Impact Factor: 3.02). 02/2003; 42(6):1377-82. DOI: 10.1021/bi027152m
Source: PubMed

ABSTRACT Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.

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Available from: Lan Guan, Sep 27, 2015
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    • "Superimposition of FucP triplet C and LacY triplet B reveals that Phe308 (helix VIII) in FucP is at exactly the same position as Trp151 in LacY (Fig. 4). An aromatic side chain at position 151 in LacY, ideally Trp, is an absolute requirement for binding and transport (Guan et al., 2003). FucP mutants F308A and F308D transport very poorly, consistent with the possible functional equivalence of Phe308 to Trp151 in LacY (Fig. 3). "
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    ABSTRACT: The major facilitator superfamily (MFS) is a diverse group of secondary transporters with members found in all kingdoms of life. The paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which has been the test bed for the development of many methods applied for the analysis of transport proteins. X-ray structures of an inward-facing conformation and the most recent structure of an almost occluded conformation confirm many conclusions from previous studies. One fundamentally important problem for understanding the mechanism of secondary active transport is the identification and physical localization of residues involved in substrate and H(+) binding. This information is exceptionally difficult to obtain with the MFS because of the broad sequence diversity among the members. The increasing number of solved MFS structures has led to the recognition of a common feature: inverted structure-repeat, formed by fused triple-helix domains with opposite orientation in the membrane. The presented method here exploits this feature to predict functionally homologous positions of known relevant positions in LacY. The triple-helix motifs are aligned in combinatorial fashion so as to detect substrate and H(+)-binding sites in symporters that transport substrates, ranging from simple ions like phosphate to more complex disaccharides. © 2015 Elsevier Inc. All rights reserved.
    Methods in Enzymology 03/2015; 557. DOI:10.1016/bs.mie.2014.12.015 · 2.09 Impact Factor
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    • "The results show that ∼70% of bound monosaccharide units show an aromatic residue interacting with the carbohydrate aliphatic core, with Trp being the predominant (43% of the cases) followed by Tyr (33%) and Phe (24%). Overall these results are consistent with previous observations from our group and others (Asensio et al. 2000; Lütteke et al. 2005; Guardia et al. 2011), which show that lectins bind their ligands combining polar interactions with the ligand –OH group and an aromatic (nonpolar) interaction with the ligand aliphatic core (Guan et al. 2003; Sujatha et al. 2004; Terraneo et al. 2007; Laughrey et al. 2008; Nishio et al. 2014). "
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    ABSTRACT: Understanding protein–ligand interactions is a fundamental question in basic biochemistry, and the role played by the solvent along this process is not yet fully understood. This fact is particularly relevant in lectins, proteins that mediate a large variety of biological processes through the recognition of specific carbohydrates. In the present work, we have thoroughly analyzed a nonredundant and well-curated set of lectin structures looking for a potential relationship between the structural water properties in the apo-structures and the corresponding protein–ligand complex structures. Our results show that solvent structure adjacent to the binding sites mimics the ligand oxygen structural framework in the resulting protein–ligand complex, allowing us to develop a predictive method using a Naive Bayes classifier. We also show how these properties can be used to improve docking predictions of lectin–carbohydrate complex structures in terms of both accuracy and precision, thus developing a solid strategy for the rational design of glycomimetic drugs. Overall our results not only contribute to the understanding of protein–ligand complexes, but also underscore the role of the water solvent in the ligand recognition process. Finally, we discuss our findings in the context of lectin specificity and ligand recognition properties.
    Glycobiology 09/2014; 25(2). DOI:10.1093/glycob/cwu102 · 3.15 Impact Factor
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    • "But replacement of Glu126 with Ala results in an increase in the BD reactivity of Arg144, consistent with the presence of a charge pair between Arg144 and Glu126 in the absence of sugar. Trp151, two turns of helix V from Arg144, also plays an important role in substrate binding (Guan et al. 2003), although mutants W151Y and W151F catalyze active lactose transport with time courses similar to the WT. Mutant W151F or W151Y binds NPG and TDG relatively poorly, although there is relatively little change in the K m of lactose transport. "
    Membrane transporter function: to structure and beyond., Edited by Ch. Ziegler, R. Kraemer, 01/2014: chapter The Life and Times of Lac Permease: Crystals Ain't Enough, but They Certainly do Help.: pages 121-158; Springer Series in Biophysics: Trasporters.
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