Journal of Molecular Biology
Volume 432, Issue 7, 27 March 2020, Pages 2099-2120
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Carbohydrate-Binding Capability and Functional Conformational Changes of AbnE, an Arabino-oligosaccharide Binding Protein

https://doi.org/10.1016/j.jmb.2020.01.041Get rights and content

Highlights

  • AbnE is a binding protein, part of the ABC importer for arabino-oligosaccharides.

  • The crystal structure of AbnE was determined with a wide series of bound ligands.

  • A complete mapping of the interactions involved in the binding site is achieved.

  • SAXS and NMA indicate two conformational states for AbnE, closed and open.

  • Analysis of the AbnE structures provides insights into the transport mechanism.

Abstract

ABC importers are membrane proteins responsible for the transport of nutrients into the cells of prokaryotes. Although the structures of ABC importers vary, all contain four conserved domains: two nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and two transmembrane domains (TMDs), which help translocate the substrate. ABC importers are also dependent on an additional protein component, a high-affinity substrate-binding protein (SBP) that specifically binds the target ligand for delivery to the appropriate ABC transporter. AbnE is a SBP belonging to the ABC importer for arabino-oligosaccharides in the Gram-positive thermophilic bacterium Geobacillus stearothermophilus. Using isothermal titration calorimetry (ITC), purified AbnE was shown to bind medium-sized arabino-oligosaccharides, in the range of arabino-triose (A3) to arabino-octaose (A8), all with Kd values in the nanomolar range. We describe herein the 3D structure of AbnE in its closed conformation in complex with a wide range of arabino-oligosaccharide substrates (A2-A8). These structures provide the basis for the detailed structural analysis of the AbnE-sugar complexes, and together with complementary quantum chemical calculations, site-specific mutagenesis, and isothermal titration calorimetry (ITC) experiments, provide detailed insights into the AbnE-substrate interactions involved. Small-angle X-ray scattering (SAXS) experiments and normal mode analysis (NMA) are used to study the conformational changes of AbnE, and these data, taken together, suggest clues regarding its binding mode to the full ABC importer.

Introduction

The ATP-Binding Cassette (ABC) transporter superfamily is a family of integral membrane proteins present in both eukaryotes and prokaryotes. These membrane proteins couple ATP binding and hydrolysis to substrate transport out of the cell (ABC exporters) and into the cell (ABC importers) [[1], [2], [3], [4]]. The importance of ABC transporters is manifested by their representation in 5% of the E. coli genome, making it the largest protein family in that organism. It is also manifested by the presence of 48 distinct ABC transporters in humans [5], some of which are involved in various diseases, such as cystic fibrosis [6], multiple drug resistance [7], Alzheimer’s disease [8], and cancer [9]. ABC exporters are found in both eukaryotes and prokaryotes, and are involved in secretion of various molecules, such as peptides, lipids, polysaccharides, proteins, toxins, and hydrophobic drugs. ABC importers have so far been found primarily in prokaryotes, mediating the uptake of a wide range of nutrients, including mono- and oligosaccharides, organic and inorganic ions, amino acids, peptides, metals, polyamine cations, opines, and vitamins [4].

Although the structures of ABC importers and exporters vary, all contain four conserved domains: two nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and two transmembrane domains (TMDs), which help translocate the substrate. Some ABC importers are also dependent on an additional protein component, a high-affinity substrate-binding protein (SBP) that specifically binds the target ligand for delivery to the appropriate ABC transporter [10]. Experimental evidences demonstrate that transporters of different types, and even of the same fold, often function through different transport mechanisms [[11], [12], [13]]. One such transport mechanism suggests that the SBP, loaded with a substrate, binds to an inward conformation of the ABC transporter, causing it to change its conformation into a pre-translocation state. ATP then binds to the NBDs, leading to the stabilization of a closed NBD dimeric state, allowing for ATP hydrolysis and conversion of the transporter back into an outward-facing conformation [5]. This state allows the SBP to release its substrate into the inner cavity of the transporter, and subsequent ATP hydrolysis to ADP and Pi converts the transporter back into the inward-facing conformation for the final release of the substrate into the cell [5].

In Gram-positive bacteria, the SBP is usually tethered onto the cell surface via covalent attachment of an N-terminal Cys residue to the lipid membrane, or by direct fusion to the corresponding transporter [4]. Although originally associated only with ABC transport systems, it has been later realized that SBPs belong also to other membrane protein systems, such as the tripartite ATP-independent periplasmic (TRAP) transporters [14], the tripartite tricarboxylate transporters (TTT) [15], three-component regulatory systems [16,17], guanylate cyclase-atrial natriuretic peptide receptors [18], ligand-gated ion channels and G-protein coupled receptors (GPCRs) [19]. The different SBPs reported to date vary in size and share very little sequence similarity, but their basic architecture appears to be generally similar. This architecture consists of two domains connected by a hinge region, with a specific ligand-binding area located at the interface between the two domains. So far, all the known SBPs have been classified into three distinct classes (classes I–III), based on sequence similarity [20], and into six distinct clusters (clusters A–F) and subclusters, based on structural similarities [19]. At this point, only two basic conformational states have been observed for SBPs, an open conformation, and a closed conformation. However, according to currently available structures, four distinct functional states have been suggested: open-unliganded, open-liganded, closed-unliganded, and closed-liganded [19]. In most of the cases, it appears that the ligand binding shifts the open-closed equilibrium toward the closed conformation, conducted by a mechanism termed “Venus Fly-trap” [21], where the two protein domains close around the bound ligand in analogy to the trapping motion of the Venus Flytrap plant. It also appears that this is the SBP conformation subsequently responsible for binding to the relevant transporter (or other protein systems) for the trigger of ligand-transport and/or signaling [5].

Geobacillus stearothermophilus is a Gram-positive, thermophilic soil bacterium, studied extensively in the past 25 years [16,[22], [23], [24], [25], [26], [27]], possessing several protein systems dedicated for the utilization of plant cell-wall polysaccharides, including the pectic polymer arabinan [16,17,23,28]. This system is among the most studied bacterial system known for the use of arabinan, and as such, presents an excellent target for both basic research and biotechnological applications. The operon dedicated to arabinan utilization in this bacterium expresses multiple proteins, all geared to sense, import, and degrade environmental arabinan and its smaller components. This integrated system includes a three-component sensing system, which is responsible for activation of the entire utilization system by sensing the presence of extracellular arabinose, transcription factors, which are responsible for regulation of gene expression of the system, and a battery of glycosyl hydrolases, which are responsible for degradation of the oligosaccharides outside and inside the bacterium. This utilization system also includes two specific ABC importers, one of which (termed AbnEFJ) was shown to be dedicated to the transport of arabino-oligosaccharides, while the other (AraEGH) was shown to be dedicated to the transport of monomeric arabinose into the cell [16]. These two transporters have their corresponding specific SBPs, AbnE, and AraE, respectively. AbnE consists of 454 amino acids, with a calculated molecular mass of 51,295 Da [16]. The protein was shown to contain a 25-amino-acids leader peptide, ending in a cysteine residue (Cys25), which is presumably attached to the lipid membrane. Using isothermal titration calorimetry (ITC), purified AbnE was shown to bind medium-sized arabino-oligosaccharides (in the range of arabino-triose (A3) to arabino-octaose (A8)), all with Kd values in the nanomolar range [16].

Here we describe the 3-D structure of AbnE in its closed conformation, in complex with a range of arabino-oligosaccharide substrates (A2-A8). These structures provide the basis for the detailed structural analysis of the physiologically-relevant AbnE-sugar complexes, and together with complementary quantum chemical calculations and isothermal titration calorimetry (ITC) experiments, provide detailed insights into the specific AbnE-substrate interactions involved. Small-angle X-ray scattering (SAXS) measurements and normal mode analysis (NMA) are used to study the conformational changes of AbnE, and all together, these data provide clues regarding its binding mode to the full ABC importer.

Section snippets

X-ray crystal structure of AbnE in complex with different arabino-oligosaccharides

The X-ray 3D crystal structure of the AbnE protein was determined to resolutions of 1.60–2.47 Å when bound to a series of seven arabino-oligosaccharides (labeled An; where n represents the number of arabinose units), ranging from arabino-biose (A2) to arabino-octaose (A8) (Table 1). In the case of the AbnE-A7 and AbnE-A8 complexes, however, it was possible to model unequivocally only six of the arabinose units of the substrate in the binding site, since the electron density of the other units

Structural basis for the binding affinity of the different arabino-oligosaccharides

In this report, we present the 3D structure of the AbnE-binding protein in complex with a series of arabino-oligosaccharides of increasing lengths, ranging from two to eight sugar units. These structures enable the detailed mapping of the specific residues involved in binding of the different oligosaccharides, and especially the identification of the exact number and types of interactions these residues form with the bound sugar substrates. These interactions include multiple hydrogen bonds, as

Cloning, overexpression, and purification of the AbnE proteins

The abnE gene was amplified via PCR, using the chromosomal DNA of G. stearothermophilus T-6 as a template. The abnE gene (GeneBank accession No. DQ868502, base pairs 22425–23789) was cloned without its first 33 amino acids, since these amino acids correspond to a leader peptide of the protein. The presence of this leader peptide appeared to be toxic for the expression system E.coli that was used. The abnE(-33AA) gene was cloned into the T7 polymerase expression vector pET9d (Novagen).

Wild-type

Acknowledgments

This work was supported by the Israel Science Foundation Grants 500/10, 152/11 and 1505/15, the Israeli Ministry of Science and Technology (MOST) Grant 3-12484, the I-CORE Program of the Planning and Budgeting Committee, the Israeli Ministry of Environmental Protection, the Israeli Ministry of Science, and the Grand Technion Energy Program (GTEP), which comprises part of The Leona M. and Harry B. Helmsley Charitable Trust reports on Alternative Energy series of the Technion, Israel Institute of

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