% IMPORTANT: The following is UTF-8 encoded.  This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.

@PHDTHESIS{Skawinska:452457,
      author       = {Skawinska, Natalia},
      title        = {{A}llosteric regulation of human tryptophan hydroxylase
                      isoform 2 (h{TPH}2)},
      school       = {Technical University of Denmark},
      type         = {Dissertation},
      reportid     = {PUBDB-2020-04692},
      pages        = {1-279},
      year         = {2020},
      note         = {Dissertation, Technical University of Denmark, 2020},
      abstract     = {Tryptophan hydroxylase catalyzes the rate-limiting reaction
                      in the biosynthesis pathway of serotonin, one of the most
                      ubiquitous and multifunctional neurotransmitters. The
                      influence of the serotonergic system extends over many
                      functions of the human body, ranging from temperature and
                      muscle tone control, to mood and sleep regulation.
                      Consequently, many diseases are associated with incorrect
                      serotonin levels, most notably major depression.Out of the
                      two isoforms of tryptophan hydroxylase expressed in the
                      human body, this thesis focuses on isoform 2 (hTPH2),
                      prevalent in the raphe nuclei of the brain. The purpose of
                      the project was the structural characterization of the
                      protein, with emphasis on its poorly understood and
                      inherently unstable regulatory domain. While the catalytic
                      and tetramerization domains of hTPH2 have been crystallized,
                      the majority of structural information about the regulatory
                      domain is inferred from the structure phenylalanine
                      hydroxylase, a close homolog of hTPH.The common thread of
                      this project was the study of allosteric ligand binding in
                      the regulatory domain of hTPH2 and its structural effects.
                      L-Phe and L-Trp have previously been reported to stabilize
                      and induce the dimerization of hTPH2 variants containing the
                      regulatory domain. Numerous biophysical techniques were
                      employed, most prominent of which were small-angle X-ray
                      scattering combined with size exclusion chromatography
                      (SEC-SAXS) and hydrogen-deuterium exchange coupled to mass
                      spectrometry (HDX-MS).A solution structure of an
                      N-terminally truncated hTPH2 tetramer in the presence of
                      0.6–9.0 mM L-Phe was successfully modeled. The resulting
                      structures are the first experimental tetrameric structures
                      of hTPH2, and the first to contain the regulatory domain of
                      the protein. The tetramer was revealed to be X-shaped and
                      inflexible, and the regulatory domains were found to be in
                      proximity to each other. The obtained structures shed new
                      light on the L-Phe bound conformation of hTPH2 and the
                      relative positions of all domains in the tetramer.A dimeric
                      hTPH2 variant lacking the tetramerization domain was also
                      modeled in the presence of L-Phe, and was found to be
                      flexible, unlike the tetramer. Modeling of this variant in
                      the presence of L-Trp was not feasible, but it was
                      nonetheless confirmed to exist in the same dimeric state in
                      the presence of L-Trp as in the presence of L-Phe. Both
                      amino acid ligands were then demonstrated to bind to the
                      same, specific site in the regulatory domain. These results
                      provide ample, multifaceted evidence for the binding of both
                      L-Phe and L-Trp at the interface of two regulatory domains,
                      which stabilizes their dimer.iiThe effects of ligand binding
                      on the structural dynamics of the hTPH2 regulatory and
                      catalytic domains were explored in a study employing
                      hydrogen-deuterium exchange coupled to mass spectrometry.
                      L-Phe binding in the active site was found to structurally
                      destabilize the C-terminal section of the catalytic domain,
                      as well as a short loop containing Thr413, a residue
                      involved in substrate selectivity of the protein. Structural
                      effects in the regulatory domain are also reported. Based on
                      strong L-Phe-induced stabilization, we were able to propose
                      the α1 and β1 elements of the regulatory domain as the
                      location of the monomer-monomer interface. This region
                      coincides with the stipulated allosteric ligand binding
                      site. Further, the interface between the catalytic and
                      regulatory domains was proposed to encompass β4 element and
                      the linker loop between the two domains.},
      cin          = {EMBL-User},
      cid          = {I:(DE-H253)EMBL-User-20120814},
      pnm          = {6G3 - PETRA III (POF3-622)},
      pid          = {G:(DE-HGF)POF3-6G3},
      experiment   = {EXP:(DE-H253)P-P12-20150101},
      typ          = {PUB:(DE-HGF)11},
      doi          = {10.3204/PUBDB-2020-04692},
      url          = {https://bib-pubdb1.desy.de/record/452457},
}