Mapping periplasmic binding protein oligosaccharide recognition with neutron crystallography

www.nature.com/scientificreports OPEN Mapping periplasmic binding protein oligosaccharide recognition with neutron crystallography Shantanu Shukla1,2,3, Dean A. Myles1* & Matthew J. Cuneo1,4* Numerous studies have shown how periplasmic binding proteins (PBPs) bind substrates with exquisite specificity, even distinguishing between sugar epimers and anomers, or structurally similar ions. Yet, marked substrate promiscuity is also a feature encoded in some PBPs. Except for three sub-Ångström crystal structures, there are no reports of hydrogen atom positions in the remaining (> 1000) PBP structures. The previous X-ray crystal structure of the maltodextrin periplasmic-binding protein from Thermotoga maritima (tmMBP) complexed with oligosaccharide showed a large network of interconnected water molecules stretching from one end of the substrate binding pocket to the other. These water molecules are positioned to form multiple hydrogen bonds, as well as forming interactions between the protein and substrate. Here we present the neutron crystal structure of tmMBP to a resolution of 2.1 Å. This is the first neutron crystal structure from the PBP superfamily and here we unambiguously identify the nature and orientation of the hydrogen bonding and watermediated interactions involved in stabilizing a tetrasaccharide in the binding site. More broadly, these results demonstrate the conserved intricate mechanisms that underlie substrate-specificity and affinity in PBPs. Periplasmic binding proteins (PBPs) are the ligand-binding components of ATP-binding cassette (ABC) transporters and are sometimes associated with chemotaxis systems. Members of the PBP superfamily mediate the uptake and transmembrane transport of a diversity of metabolically important solutes in bacteria, such as carbohydrates, ions, amino acids, and polyamines, to name a few. Despite the wide variation in PBP ligand size and chemical functionality the two domain PBPs have a conserved αβ-fold, with each domain encompassing a central β-stranded core that is sandwiched between surrounding α-helices1. Ligand binding at the interdomain interface induces a hinge-bending motion in which the two solvent exposed polar interfaces of each domain close down on and envelop ligands in a largely desolvated binding site reminiscent of the protein interior2–4. However, in many instances water molecules remain within the binding site, and these solvent interactions with ligands have been shown to be important in broadening PBP substrate specificity or fine tuning highly specific b inding5,6. For example, in PBPs that share the oligopeptidebinding protein fold, water molecules have been shown to act as adapters, matching the ligand hydrogen-bonding requirements and permitting a diverse array of substrates to be accommodated within a single binding s ite7,8. In some cases, rather than serving to increase promiscuity, water molecules are also used in PBPs to impart specificity beyond what can be encoded within the repertoire of protein backbone and side-chains6,9. In yet others, PBPs have been shown to have bipartite binding sites, where each sub-site differentially uses water to impart either specificity or promiscuity2,10. Taken together, the diversity of PBP binding site adaptation mechanisms suggests that determining water molecule orientation and hydrogen bonding patterns within the PBP substrate binding site is indispensable for understanding the fine tuning of ligand recognition within this class of proteins. More than 1000 crystal structures of PBPs have been determined thus far. Of these, only three were determined at sub-Ångstrom resolution; glucose-binding protein (PDB ID:2FVY)11, glutamine-binding protein (PDB ID: 4KQP)12, and a phosphate-binding protein (PDB ID: 4F1V)13. In these three crystal structures, the resolution of the diffraction data permitted the explicit positioning of many protein hydrogen atoms, yet no hydrogen atoms could be modeled on solvent molecules. In all others, the position of key hydrogen atoms and patterns of hydrogen bonding interactions within the PBP binding site are inferred, rather than experimentally determined. 1 Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. 2UT‑ORNL Graduate School in Genome Science and Technology, The University of Tennessee at Knoxville, Knoxville, TN 37966, USA. 3Department of Microbiology and Immunology, Northwestern University, Chicago, IL 60611, USA. 4Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA. *email: ; Scientific Reports | (2022) 12:17647 | https://doi.org/10.1038/s41598-022-20542-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ Here we report the first neutron crystal structure of a PBP, namely a maltodextrin-binding protein (MBP) from a cluster of three related proteins found in Thermotoga maritima (tmMBP)14. This study reveals the explicit hydrogen bonding interactions and water network within the substrate binding site at an unprecedented level of detail, lending valuable insights into the intricate mechanisms that underpin the derivation of substratespecificity and affinity in PBPs. Results Neutron structure of maltotetraose bound tmMBP. The genome of T. maritma encodes for three isoforms of maltose binding proteins; the tmMBP2 isoform was used in the present study and is called tmMBP going forwards. Large crystals of tmMBP that were suitable for neutron diffraction experiments were grown in sitting drops using nine-well siliconized glass plates. Each sitting drop was made by mixing 400 μl of protein with an equal volume of the mother liquor supplemented with 10% (vol./vol.) deuterated glycerol to slow the rate of crystallization and growth, and incubated at 20 °C. Large crystals (3–10 mm3) appeared within 30 days and were transferred weekly into sandwich boxes with fresh deuterated mother liquor to promote replacement of exchangeable hydrogen atoms for deuterium. A total of four mother liquor exchanges were performed. Crystals were transferred to quartz capillaries with a D 2O mother liquor plug and sealed with wax for room temperature neutron and X-ray data collection. The room temperature X-ray crystal structure of the tmMBP complexed with maltotetraose was determined at 1.70 Å with an R work/Rfree of 14.1/17.5%. The X-ray model contained a total of 3099 non-hydrogen atoms (C, N, O, S), of which 2893 were protein atoms, 45 ligand atoms, and 161 solvent atoms. Prior to joint refinement, hydrogen atoms were added to the structure, where exchangeable positions had mixed hydrogen/deuterium occupancy. This starting model was used for joint X-ray/neutron refinement in P henix15,16 where H/D occupancy and position was experimentally modelled based on the neutron diffraction data. The final room temperature neutron structure was determined to a resolution of 2.10 Å and refined to an Rwork/Rfree of 24.3/27.3%. Data collection statistics and details on the (...truncated)