Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

Abstract

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of ␤(134)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.

Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae

“…Carbohydrate utilization by T. maritima has been examined by studying the substrate specificities and affinities of its carbohydrate transporters (Nanavati et al 2005(Nanavati et al , 2006Cuneo et al 2009;Boucher and Noll 2011;Ghimire-Rijal et al 2014) and their transcriptional regulation in response to growth on different saccharides (Frock et al 2012). Information about substrate specificities, enzymatic activities, and catalytic mechanisms of many of T. maritima's glycoside hydrolases are also available (Kleine and Liebl 2006;Comfort et al 2007;Arti et al 2012), which has been used, for instance, to engineer an ␣-galactosidase from T. maritima into an efficient ␣-galactosynthase (Cobucci-Ponzano et al 2011).…”

Duplication of Genes in an ATP-binding Cassette Transport System Increases Dynamic Range While Maintaining Ligand Specificity

“…Like other PBPs with the oligopeptide binding fold, the tmMnBP proteins are composed of three domains with domains I and II forming the N-terminal half of the ligand binding site and domain III forming the C-terminal half. The ordering of ␤-strands in domains I and III places tmMnBP3 and tmMnBP6 in the Group II PBP subfamily (29) or in the C cluster based on a more recent, updated classification of PBPs (30). In both of these classification systems, the closest structural homolog, the T. maritima cellobiose-binding protein (tmCBP) is also found.…”

Adaptive Evolution of Thermotoga maritima Reveals Plasticity of the ABC Transporter Network

“…This transporter recognizes and imports beta-glucosides such as cellobiose. However, this mutation to bglE is near the center of the gene and eliminates key tryptophan residues (W381, W384, and W536) responsible for forming van der Waals interactions with cellobiose (53).…”

“…This is the result of a chromosomal gene deletion to Tmari_0030, but, more importantly, the promoter governing expression of the operon carrying bglEFGKL, bglR, and other pathway genes is also eliminated. The third evolved culture harbors a nonsense mutation that truncates key amino acids necessary for binding of beta-glucosides (53). These changes to the functionality of BglEFGKL reveal a potential regulatory inefficiency due to effector overlap between GluR and BglR.…”

“…These changes to the functionality of BglEFGKL reveal a potential regulatory inefficiency due to effector overlap between GluR and BglR. Glucose induces a strong transcriptional response for bglEFGKL, which is known to transport only beta-glucoside polymers comprised of 2 to 5 monomers (53,54,61). Therefore, while GluR senses glucose and induces a beneficial transcriptional response, BglR activation by glucose produces a greater transcriptional response that is ineffectual in the uptake of glucose monomers.…”