Abstract
BackgroundMembers of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to specifically bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. The tmCBP binding site is bipartite, comprising a canonical solvent-excluded region (subsite one), adjacent to a solvent-filled cavity (subsite two) where specific and semi-specific ligand recognition occur, respectively.ResultsA molecular level understanding of binding pocket adaptation mechanisms that simultaneously allow both ligand specificity at subsite one and promiscuity at subsite two has potentially important implications in ligand binding and drug design studies. We sought to investigate the determinants of ligand binding selectivity in tmCBP through biophysical characterization of tmCBP in the presence of varying β-glucan oligosaccharides. Crystal structures show that whilst the amino acids that comprise both the tmCBP subsite one and subsite two binding sites remain fixed in conformation regardless of which ligands are present, the rich hydrogen bonding potential of water molecules may facilitate the ordering and the plasticity of this unique PBP binding site.ConclusionsThe identification of the roles these water molecules play in ligand recognition suggests potential mechanisms that can be utilized to adapt a single ligand binding site to recognize multiple distinct ligands.
Highlights
Members of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to bind a chemically diverse range of ligands
Addition of LR2 and LR5 shifted the thermal melting point (Tm) of the protein from 94.8°C to 99.2°C and 105.2°C respectively (Figure 2). These studies indicate that the Thermotoga maritima cellobiose-binding protein (tmCBP) binding site accommodates β(1,3) glucosaccharides ranging in size from two to five sugar rings, which is consistent with earlier circular dichroism (CD) binding studies for the β(1,4) glucosaccharides cellobiose and cellopentaose [13]
Depending on biological function, PBPs ligand selectivity is modulated through a combination of binding pocket adaptations that mediate ligand positioning, alter ligand size selection, or alter the free energy of ligand binding in such a manner that excludes incorrect ligands [35,40]
Summary
Members of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. Despite the wide variation in PBP cognate ligand size and chemical functionality, the three-dimensional structure is highly conserved across all PBPs. PBPs are comprised of two α/β domains connected by a flexible linker region that serves as a pivot point for the ligand induced hinge-bending motion that this protein superfamily is known for [17,18,19,20]. The PBP super-family was re-categorized into six distinct clusters by combining known ligand specificities with the wealth of structural information available in the Protein Data Bank [22]
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