Dissertation committee:
Γεώργιος Διαλλινάς, Καθηγητής, ΕΚΠΑ
Bruno André, Καθηγητής, Université Libre de Bruxelles
Εμμανουήλ Μικρός, Καθηγητής, ΕΚΠΑ
Κωνσταντίνος Βοργιάς, Καθηγητής, ΕΚΠΑ
Δημήτρης Χατζηνικολάου, Καθηγητής, ΕΚΠΑ
Ευστάθιος Φριλίγγος, Καθηγητής, Πανεπιστήμιο Ιωαννίνων
Χρήστος Γουρνάς, Ερευνητής Γ', ΕΚΕΦΕ 'Δημόκριτος'
Summary:
Transporters are membrane proteins that mediate the import and export of nutrients,
metabolites, signaling molecules or drugs in and out of cells, and thus are essential for their
communication with the environment. In the recent years, genetic, biochemical and biophysical data
for several transporters have come to provide us with new knowledge concerning structure-function
relationships and mechanisms of substrate recognition and transport. Despite their evolutionary,
structural and functional differences all transporters use an alternating-access mechanism where a
substrate binding site, in allosteric co-operation with gating domains, alternates between multiple
conformations for receiving and delivering specific substrate(s) from one side of the membrane to
the other. This basic mechanism, carried out by dynamic movements of the main transmembrane
body and assisted by the flexibility of interconnecting hydrophilic loops, exists in different variations,
known as the rocker-switch, the rocking-bundle or the elevator sliding mechanisms.
One of the largest families of transporters is the amino acid/polyamine/organocation (APC)
superfamily, which includes members that function as solute:cation symporters and solute:solute
antiporters with varying specificities. The Nucleobase Cation Symporter 1 (NCS1) family consists one
of the best-studied subfamilies of the APC superfamily and is present in prokaryotes, fungi and some
plants. This is due to a plethora of genetic and biochemical findings concerning fungal members of
the family, as well as, extensive structural and biophysical data concerning a bacterial homologue,
namely the benzyl-hydantoin/Na+
Mhp1 symporter. Absence of NCS1 homologues in mammals
makes this family an ideal, highly specific gateway to target nucleobase-specific drugs to microbial
pathogens. NCS1 proteins consist of 12 transmembrane α-helical segments (TMS) interconnected
with rather short loops and cytosolic N- and C-termini. TMSs 1-10 are arranged as a 5-helix
intertwined inverted repeat (5HIRT), the so called LeuT-fold, also found in different transporter
families involved in neurotransmitter, sugar, amino acid or drug transport. The last two TMSs (11 and
12) in all LeuT-like proteins seem to be crucial for the oligomerization state of some NCS1-similar
transporters, rather than being involved in the mechanism of transport. However, formal evidence
for their structural and/or functional role is missing.
Fungal NCS1 proteins are among the best-studied transporters at a genetic, biochemical and
cellular level. Structural models of fungal NCS1 transporters, based on several distinct crystal
structures of the bacterial homologue Mhp1 are fully supported by genetic studies that identify the
substrate binding site and putative gating elements determining specificity. More specifically, a major
novel finding that has originated from our work on fungal NCS1 transporters (both on Fur and Fcy
ix
members) is that substrate specificity is determined not only by residues of the substrate binding site
(TMS1, TMS3, TMS6 or TMS8), but also by dynamic movements of the TMS9-TMS10 region, acting as
an outward-facing gate.
In work described herein, we provide experimental and in silico evidence that the turnover,
function, and interestingly, the specificity of an Aspergillus nidulans NCS1 homologue, namely the
FurE uracil-allantoin-uric acid transporter, depends on dynamic interactions of the N- and C-terminal
cytoplasmic regions with each other and the main body of the transporter. We specifically show that
the N- and C-terminal domains of FurE are involved in intramolecular dynamics critical for the fine
regulation of the mechanism of gating that controls substrate selection. Using Molecular Dynamics
(MD) and mutational analysis we postulate that this occurs via interactions of the cytoplasmic tails
with the cytoplasmic loops, which in turn affect the gating process at the extracellular side of the
plasma membrane (PM) in a pH-dependent manner.
Next, using the information from extensive MD analysis, we performed a targeted systematic
mutational and functional analysis to characterize the substrate translocation trajectory in FurE,
during its transition from the outward-open to the inward-open conformation. Additionally, we
provide experimental evidence that the nature of the amino acid residues of the last two
transmembrane domains of FurE is not critical for the transport catalysis. However, a single tyrosine
residue is absolutely necessary for the proper trafficking of the transporter to the plasma membrane.
Finally, in the last part of this work we wanted to combine biophysical techniques and
computational modeling in order to better understand the mechanism underlying the functional role
of cytoplasmic tails in substrate selection and translocation, which seems to reflect a more general
mechanism that controls APC transporters. In general, our work supports the emerging concept that
the size of eukaryotic transporter termini increased during evolution and adds more and different
modes of regulation.
Keywords:
Aspergillus nidulans, fungi, genetics, gating, transport, folding, cytoplasmic termini, substrate, specificity
