The 3D structure of the Na+-hydantoin transport protein, Mhp1, in different conformational states had already been established. To elucidate the molecular mechanisms of Mhp 1 these structural data needed support from functional data. The aim of this project was to characterise functionally Mhp 1 and to combine these data with structural information to illuminate the mechanisms of ligand binding and transport. This potentially gives insights into the molecular mechanisms of structurally similar transporters of therapeutic importance, which are reviewed. The wild-type Mhp1 protein, sixteen proteins mutated in the ligand binding site and six proteins mutated in the sodium binding site of Mhpl were expressed and purified prior to biochemical and biophysical analyses. The sodium-dependent binding of L-benzylhydantoin (L-BH) to the purified proteins was investigated using spectrophototluorimetry. The uptake of radiolabelled L-indolylmethylhydantoin (L-IMH) by the Mhpl proteins expressed in whole cells was also assessed. The mutation of Gln42, Trpl17, Gln121, Gly219, Trp220 or Asn318 in the ligand binding site impaired severely ligand binding and/or transport, revealing important interactions that these residues make with the bound ligand. The mutation of Ala317 in the ligand binding site or Ser312 or Thr313 in the sodium binding site removed the sodium-dependence of ligand binding and the Ala309Asn mutant exhibited a sodium-independent high ligand binding affinity. The mutation of Met39, Ile41, Gln153, Ala222 or Asn314 had significant but less dramatic impacts. Analyses of all of the mutant phenotypes lead to proposals of how the ligand and sodium binding sites might be linked. A library of seventy five compounds was designed to probe the recognition of ligands by Mhpl and these were screened for their ability to inhibit radiolabelled L-IMH uptake by the wild-type protein. The hydantoin moiety and a hydrophobic R group attached at the 5-postion of the hydantoin were shown to be crucial in generating compounds with a reasonable binding affinity. Five compounds, DIL naphthylmethylhydantoin (DIL-NMH), D-NMH, L-NMH, bromovinylhydantoin (BVH) and iodovinylhydantoin (IVH), were discovered that were more potent than the previously identified highest affinity ligand, L- IMH. The binding of L-NMH to the purified wild-type protein was investigated by spectrophotofluorimetry and near UV synchrotron radiation CD (SRCD). The lack of significant transport of radiolabelled L-NMH by cells expressing Mhpl suggested that L- NMH may act as a competitive inhibitor. A crystal structure ofD/L-NMH solved in complex with wild-type Mhpl, by collaborators, showed that the extracellular helix 10 'thin' gate did not occlude the ligand binding site fully, suggesting that closure of the extracellular 'thin' gate may be a prerequisite for larger conformational changes to convert Mhp1 from an outward- facing to an inward-facing structure.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589026 |
| Date | January 2012 |
| Creators | Jackson, Scott Michael |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.002 seconds