RESUMO
In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Espectroscopia de Ressonância Magnética , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Flúor , Histidina/química , Modelos Moleculares , Mutação , Níquel/química , Conformação Proteica , Domínios Proteicos , Pyrococcus horikoshii/químicaRESUMO
Cancer cells undergo a shift in metabolism where they become reliant on nutrients such as the amino-acid glutamine. Glutamine enters the cell via the alanine/serine/cysteine transporter 2 (ASCT2) that is upregulated in several cancers to maintain an increased supply of this nutrient and are therefore an attractive target in cancer therapeutic development. ASCT2 belongs to the glutamate transporter (SLC1A) family but is the only transporter in this family able to transport glutamine. The structural basis for glutamine selectivity of ASCT2 is unknown. Here, we identify two amino-acid residues in the substrate-binding site that are responsible for conferring glutamine selectivity. We introduce corresponding mutations into a prokaryotic homologue of ASCT2 and solve four crystal structures, which reveal the structural basis for neutral amino acid and inhibitor binding in this family. This structural model of ASCT2 may provide a basis for future development of selective ASCT2 inhibitors to treat glutamine-dependent cancers.
Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Glutamina/metabolismo , Sequência de Aminoácidos , Aminoácidos , Animais , Sítios de Ligação , Escherichia coli , Mutagênese Sítio-Dirigida , Neoplasias/metabolismo , Oócitos , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Especificidade por Substrato , Xenopus laevisRESUMO
Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation.
Assuntos
Sequência de Aminoácidos , Membrana Celular/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Dobramento de Proteína , Deleção de Sequência , Animais , Membrana Celular/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Humanos , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Xenopus laevisRESUMO
Glutamate transporters are integral membrane proteins that catalyse a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, respectively. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homologue from Pyrococcus horikoshii, Glt(Ph), which is trapped in the inward facing state by cysteine crosslinking. Together with the previously determined crystal structures of Glt(Ph) in the outward facing state, the structure of the crosslinked mutant allows us to propose a molecular mechanism by which Glt(Ph) and, by analogy, mammalian glutamate transporters mediate sodium-coupled substrate uptake.