Allosteric regulation of human tryptophan hydroxylase isoform 2 (hTPH2)

Natalia Teresa Skawinska*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesisResearch

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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.
ii
The 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.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages279
Publication statusPublished - 2020

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