The membrane-associated lipoprotein-9 GmpC from Staphylococcus aureus binds the dipeptide GlyMet via side chain interactions

Bacterial dipeptide ABC transporters function to import a wide range of dipeptide substrates. This ability to transport a wide variety of dipeptides is conferred by the cognate substrate binding protein (SBP) of these transporters. SBPs bind dipeptides with little regard for their amino acid content. Here, we report the 1.7 A resolution structure of lipoprotein-9 (SA0422) of Staphylococcus aureus in complex with the dipeptide glycylmethionine. Experimental characterization of the subcellular location of the protein confirmed that SA0422 is an acylated, peripheral membrane protein. This is the first structure determined for an SBP of a Gram-positive dipeptide ABC transporter. Usually, binding of dipeptides occurs in a binding pocket that is largely hydrated and able to accommodate the side chains of several different amino acid residues. Unlike any other known SBP, lipoprotein-9 binds the side chains of the glycylmethionine dipeptide through very specific interactions. Lipoprotein-9 shares significant structural and sequence homology with the MetQ family of methionine SBP. Sequence comparisons between MetQ-like proteins and lipoprotein-9 suggest that the residues forming the tight interactions with the methionine side chains of the ligand are highly conserved between lipoprotein-9 and MetQ homologues, while the residues involved in coordinating the glycine residue are not. Modeling of the Vibrio cholerae MetQ and lipoprotein-9 binding pockets can account for lipoprotein-9 substrate specificity toward glycylmethionine. For this reason, we have designated lipoprotein-9 GmpC, for glycylmethionine binding protein.     

A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF).

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3'-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA(Sec) is not recognized by elongation factor EF-1alpha, but is recognized specifically by a Sec-tRNA(Sec) protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA(Sec), providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.     

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Identification of various substrate-binding proteins of the hyperthermophylic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> K1

Proteins from the extracellular medium of Aeropyrum pernix K1 were separated by two-dimensional electrophoresis and identified using mass spectrometry. Six different substrate-binding proteins (SBPs) from the ATP-binding cassette (ABC) transporter family were identified: (1) ABC transporter SBP (Q9YC61); (2) Branched-chain amino-acid ABC transporter, branched-chain amino-acid-binding protein (Q9YDJ6); (3) Oligopeptide ABC transporter, oligopeptide-binding protein (Q9YBL5); (4) Probable ABC transporter SBP (Q9Y9N4); (5) ABC transporter SBP (Q9YBG7); (6) ABC transporter SBP (Q9YFD7). Based on their orthology, division into the following classes was predicted: (1) multiple sugar-transport system SBPs; (2) peptide/nickel-transport system SBPs; and (3) branched-chain amino-acid-transport system SBPs. Further bioinformatic analyses showed that the identified SBPs differ in motif and in transmembrane-domain and signal-peptide organisation. Additionally, for all of these SBPs, sequence homology was found for archaeal proteins, and homologous proteins in bacteria were also found for the ABC transporter SBP Q9YBG7 and the ABC transporter SBP Q9YFD7. This is the first study, where different ABC SBPs from the extracellular medium of A. pernix have been identified using the combined methodology of two-dimensional electrophoresis and mass spectrometry.     

Crystal structures of three representatives of a new Pfam family PF14869 (DUF4488) suggest they function in sugar binding/uptake

Crystal structures of three members (BACOVA_00364 from Bacteroides ovatus, BACUNI_03039 from Bacteroides uniformis and BACEGG_00036 from Bacteroides eggerthii) of the Pfam domain of unknown function (DUF4488) were determined to 1.95, 1.66, and 1.81 Å resolutions, respectively. The protein structures adopt an eight-stranded, calycin-like, β-barrel fold and bind an endogenous unknown ligand at one end of the β-barrel. The amino acids interacting with the ligand are not conserved in any other protein of known structure with this particular fold. The size and chemical environment of the bound ligand suggest binding or transport of a small polar molecule(s) as a potential function for these proteins. These are the first structural representatives of a newly defined PF14869 (DUF4488) Pfam family.     

The size distribution of protein families within different types of folds

It is well known that the structure is currently available only for a small fraction of known protein sequences. It is urgent to discover the important features of known protein sequences based on present protein structures. Here, we report a study on the size distribution of protein families within different types of folds. The fold of a protein means the global arrangement of its main secondary structures, both in terms of their relative orientations and their topological connections, which specify a certain biochemical and biophysical aspect. We first search protein families in the structural database SCOP against the sequence-based database Pfam, and acquire a pool of corresponding Pfam families whose structures can be deemed as known. This pool of Pfam families is called the sample space for short. Then the size distributions of protein families involving the sample space, the Pfam database and the SCOP database are obtained. The results indicate that the size distributions of protein families under different kinds of folds abide by similar power-law. Specially, the largest families scatter evenly in different kinds of folds. This may help better understand the relationship of protein sequence, structure and function. We also show that the total of proteins with known structures can be considered a random sample from the whole space of protein sequences, which is an essential but unsettled assumption for related predictions, such as, estimating the number of protein folds in nature. Finally we conclude that about 2957 folds are needed to cover the total Pfam families by a simple method.     

Structural basis for the binding of compatible solutes by ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus

Compatible solutes such as glycine betaine and proline betaine serve as protein stabilizers because of their preferential exclusion from protein surfaces. To use extracellular sources of this class of compounds as osmo-, cryo-, or thermoprotectants, Bacteria and Archaea have developed high affinity uptake systems of the ATP-binding cassette type. These transport systems require periplasmic- or extracellular-binding proteins that are able to bind the transported substance with high affinity. Therefore, binding proteins that bind compatible solutes have to avoid the exclusion of their ligands within the binding pocket. In the present study we addressed the question to how compatible solutes can be effectively bound by a protein at temperatures around 83 degrees C as this is done by the ligand-binding protein ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus. We solved the structures of ProX without ligand and in complex with both of its natural ligands glycine betaine and proline betaine, as well as in complex with the artificial ligand trimethylammonium. Cation-pi interactions and non-classical hydrogen bonds between four tyrosine residues, a main chain carbonyl oxygen, and the ligand have been identified to be the key determinants in binding the quaternary amines of the three investigated ligands. The comparison of the ligand binding sites of ProX from A. fulgidus and the recently solved structure of ProX from Escherichia coli revealed a very similar solution for the problem of compatible solute binding, although both proteins share only a low degree of sequence identity. The residues involved in ligand binding are functionally equivalent but not conserved in the primary sequence.     

A novel signal transduction protein: Combination of solute binding and tandem PAS‐like sensor domains in one polypeptide chain

We report the structural and biochemical characterization of a novel periplasmic ligand‐binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N‐terminal and a tandem PAS‐like sensor domain at the C‐terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS‐like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS‐like domain of the tandem PAS‐like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS‐like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non‐redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.     

Computer-based screening of functional conformers of proteins

A long-standing goal in biology is to establish the link between function, structure, and dynamics of proteins. Considering that protein function at the molecular level is understood by the ability of proteins to bind to other molecules, the limited structural data of proteins in association with other bio-molecules represents a major hurdle to understanding protein function at the structural level. Recent reports show that protein function can be linked to protein structure and dynamics through network centrality analysis, suggesting that the structures of proteins bound to natural ligands may be inferred computationally. In the present work, a new method is described to discriminate protein conformations relevant to the specific recognition of a ligand. The method relies on a scoring system that matches critical residues with central residues in different structures of a given protein. Central residues are the most traversed residues with the same frequency in networks derived from protein structures. We tested our method in a set of 24 different proteins and more than 260,000 structures of these in the absence of a ligand or bound to it. To illustrate the usefulness of our method in the study of the structure/dynamics/function relationship of proteins, we analyzed mutants of the yeast TATA-binding protein with impaired DNA binding. Our results indicate that critical residues for an interaction are preferentially found as central residues of protein structures in complex with a ligand. Thus, our scoring system effectively distinguishes protein conformations relevant to the function of interest.     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.     

Several archaeal homologs of putative oligopeptide-binding proteins encoded by Thermotoga maritima bind sugars

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the beta-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), beta-1,4-mannobiose; and TM1226 (ManD), beta-1,4-mannobiose, beta-1,4-mannotriose, beta-1,4-mannotetraose, beta-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), alpha-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.     

Structural, bioinformatic, and in vivo analyses of two Treponema pallidum lipoproteins reveal a unique TRAP transporter

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of "tetratricopeptide repeat" (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).     

Bile acid interactions with rabbit ileal lipid binding protein and an engineered helixless variant reveal novel ligand binding properties of a versatile beta-clam shell protein scaffold

The intracellular ileal lipid binding proteins (ILBPs) are involved in the transport and enterohepatic circulation of bile acids. ILBPs from different species show high sequence and structural homology and have been shown to bind multiple bile acid ligands with differing degrees of selectivity and positive co-operativity. Human ILBP binds bile acid derivatives in a well-characterised 2:1 ligand:protein complex, however, we show that the highly homologous rabbit ILBP (82% sequence identity) with seven conservative substitutions preferentially binds multiple conjugated deoxycholate ligands in a novel 3:1 binding mode essentially within the same beta-clam shell structure. We have extended these studies to investigate the role of the alpha-helical capping motif (residues 9-35) in controlling the dimensions of the binding cavity and ligand uptake. Substituting the alpha-helical motif (residues 9-35) with a short Gly-Gly-Ser-Gly linker dramatically affects the protein stability such that under physiological conditions the mutant (Deltaalpha-ILBP) is highly disordered. However, we show that the inability of the mutant to adopt a stable three-dimensional structure under these conditions is no barrier to binding ligands with near-native affinity. These structural modifications not only demonstrate the possibility of strong coupling between ligand binding and protein folding, but result in changes in bile acid selectivity and binding stoichiometry, which we characterise in detail using isothermal calorimetry and mass spectrometry.     

Implications of structural genomics target selection strategies: Pfam5000, whole genome, and random approaches

Structural genomics is an international effort to determine the three-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins should be selected according to a strategy that is medically and biologically relevant, of good value, and tractable. As an option to consider, we present the "Pfam5000" strategy, which involves selecting the 5000 most important families from the Pfam database as sources for targets. We compare the Pfam5000 strategy to several other proposed strategies that would require similar numbers of targets. These strategies include complete solution of several small to moderately sized bacterial proteomes, partial coverage of the human proteome, and random selection of approximately 5000 targets from sequenced genomes. We measure the impact that successful implementation of these strategies would have upon structural interpretation of the proteins in Swiss-Prot, TrEMBL, and 131 complete proteomes (including 10 of eukaryotes) from the Proteome Analysis database at the European Bioinformatics Institute (EBI). Solving the structures of proteins from the 5000 largest Pfam families would allow accurate fold assignment for approximately 68% of all prokaryotic proteins (covering 59% of residues) and 61% of eukaryotic proteins (40% of residues). More fine-grained coverage that would allow accurate modeling of these proteins would require an order of magnitude more targets. The Pfam5000 strategy may be modified in several ways, for example, to focus on larger families, bacterial sequences, or eukaryotic sequences; as long as secondary consideration is given to large families within Pfam, coverage results vary only slightly. In contrast, focusing structural genomics on a single tractable genome would have only a limited impact in structural knowledge of other proteomes: A significant fraction (about 30-40% of the proteins and 40-60% of the residues) of each proteome is classified in small families, which may have little overlap with other species of interest. Random selection of targets from one or more genomes is similar to the Pfam5000 strategy in that proteins from larger families are more likely to be chosen, but substantial effort would be spent on small families.     

The Overlap of Small Molecule and Protein Binding Sites within Families of Protein Structures

Protein–protein interactions are challenging targets for modulation by small molecules. Here, we propose an approach that harnesses the increasing structural coverage of protein complexes to identify small molecules that may target protein interactions. Specifically, we identify ligand and protein binding sites that overlap upon alignment of homologous proteins. Of the 2,619 protein structure families observed to bind proteins, 1,028 also bind small molecules (250–1000 Da), and 197 exhibit a statistically significant (p<0.01) overlap between ligand and protein binding positions. These “bi-functional positions”, which bind both ligands and proteins, are particularly enriched in tyrosine and tryptophan residues, similar to “energetic hotspots” described previously, and are significantly less conserved than mono-functional and solvent exposed positions. Homology transfer identifies ligands whose binding sites overlap at least 20% of the protein interface for 35% of domain–domain and 45% of domain–peptide mediated interactions. The analysis recovered known small-molecule modulators of protein interactions as well as predicted new interaction targets based on the sequence similarity of ligand binding sites. We illustrate the predictive utility of the method by suggesting structural mechanisms for the effects of sanglifehrin A on HIV virion production, bepridil on the cellular entry of anthrax edema factor, and fusicoccin on vertebrate developmental pathways. The results, available at http://pibase.janelia.org, represent a comprehensive collection of structurally characterized modulators of protein interactions, and suggest that homologous structures are a useful resource for the rational design of interaction modulators.     

SUPFAM—a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes

Members of a superfamily of proteins could result from divergent evolution of homologues with insignificant similarity in the amino acid sequences. A superfamily relationship is detected commonly after the three-dimensional structures of the proteins are determined using X-ray analysis or NMR. The SUPFAM database described here relates two homologous protein families in a multiple sequence alignment database of either known or unknown structure. The present release (1.1), which is the first version of the SUPFAM database, has been derived by analysing Pfam, which is one of the commonly used databases of multiple sequence alignments of homologous proteins. The first step in establishing SUPFAM is to relate Pfam families with the families in PALI, which is an alignment database of homologous proteins of known structure that is derived largely from SCOP. The second step involves relating Pfam families which could not be associated reliably with a protein superfamily of known structure. The profile matching procedure, IMPALA, has been used in these steps. The first step resulted in identification of 1280 Pfam families (out of 2697, i.e. 47%) which are related, either by close homologous connection to a SCOP family or by distant relationship to a SCOP family, potentially forming new superfamily connections. Using the profiles of 1417 Pfam families with apparently no structural information, an all-against-all comparison involving a sequence-profile match using IMPALA resulted in clustering of 67 homologous protein families of Pfam into 28 potential new superfamilies. Expansion of groups of related proteins of yet unknown structural information, as proposed in SUPFAM, should help in identifying ‘priority proteins’ for structure determination in structural genomics initiatives to expand the coverage of structural information in the protein sequence space. For example, we could assign 858 distinct Pfam domains in 2203 of the gene products in the genome of Mycobacterium tubercolosis. Fifty-one of these Pfam families of unknown structure could be clustered into 17 potentially new superfamilies forming good targets for structural genomics. SUPFAM database can be accessed at http://pauling.mbu.iisc.ernet.in/~supfam.     

Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded by Thermotoga maritima Bind Sugars

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the β-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), β-1,4-mannobiose; and TM1226 (ManD), β-1,4-mannobiose, β-1,4-mannotriose, β-1,4-mannotetraose, β-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), α-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.     

Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino‐acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.