BasC K154 role on cytosolic gate closing. The bacterial alanine-serine-cysteine transporter as a model

Abstract

[eng] L-type amino acid transporters play key roles in human physiology and are involved in several human pathologies such as primary inherited aminoacidurias (Feliubadaló et al., 1999; Torrents et al., 1999), autism spectrum disease (Tărlungeanu et al., 2016) and age-related hearing loss (Espino Guarch et al., 2018) among others. However, their complete and detailed transport mechanism is still unknown as the structures solved until now are most of them in the same conformational state, open-to-in, with an exception of LAT1 bound to inhibitors that showed for the first time an inward occluded conformation (Yan et al., 2021). To decipher LATs transport mechanism we have studied the bacterial LAT alanine- serine-cysteine exchanger BasC as a LAT model. With the objective to use BasC to translate from the already well described transport mechanism of the neurotransmitter sodium symporter (NSS) family, which shares the same APC fold from the APC superfamily, to the current LAT transport mechanism in study here. BasC has been already characterized (Bartoccioni et al., 2019) and 3D structurally solved in complex with the specific nanobody 74 (Errasti-Murugarren et al., 2019). Here, the 29 nanobodies have been characterised for the following parameters. Firstly, nanobodies have been characterised by their binding to sort them by affinity through Surface Plasmon Resonance. Then, by their inhibition profile to unveil their side of interaction through inhibition screenings of BasC transport activity. Also, by their recognised conformation compatibilities between them through the feasibility of tertiary complex between BasC and nanobody pairs. Finally, for their possible conformational effect on BasC through single-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements on TM1a on the cytoplasmic gate. In this regard, selected nanobodies could eventually force BasC to adopt these new conformations currently unavailable and solve them in complex through structural techniques such as X-ray crystallography and cryo-EM. Here we present two low-resolution structures of BasC-Nb71 and BasC-Nb53-58 complexes. Furthermore, the dynamics of this cytoplasmic gate were also studied under the effect of substrates (L- alanine and L-serine) and inhibitors (L-glutamine). It was detected an inward closing of the TM1a due to L-alanine and L-Serine but not due to the addition of L-glutamine, that did not provoke any closing effect by the cytosolic side. Even more, nanobodies became here a powerful tool to study the blocking effect of these substrate-induced effects. It was identified nanobody 71 as a strong blocker of it while nanobody 53 was not impeding this TM1a bend. These substrate-induced dynamic effects being blocked or not by nanobodies in combination with the two structures solved, sustains the designed smFRET approach, as the structure interactions between the nanobodies 71 and 53 towards BasC structurally explain the blocking effects of the TM1a dynamic results obtained by smFRET. Regarding the mechanism of transport of LATs, the seen substrate-induced closing effect of the cytoplasmic gate for the BasC WT variant has been studied on lysinuric protein intolerance (LPI) related variant (K154A). This Lys residue is highly conserved among all hLATs, located in the TM5 in a homologue region of the key Na2 site for the NSS family of the APC superfamily. It has been speculated the role of this conserved Lys to be one of the key factors for the sidedness of LATs and mainly responsible for the occlusion and opening of the thick gate in the cytosol. Here we obtained the first experimental evidence of the molecular mechanism deficiency when there lacks this Lys as the LPI related variant was unable to close the cytosolic gate. Overall, we present a useful approach that can be used to study the transport mechanism of membrane proteins and the miss functions of disease related variants.

[cat] Els transportadors d’L-aminoàcids (LATs) tenen un paper capital en la fisiologia humana i estan involucrats en diverses patologies com les aminoacidúries primàries heretades (Feliubadaló et al., 1999; Torrents et al., 1999), l’espectre autista (Tărlungeanu et al., 2016) o la pèrdua d’oïda relacionada amb l’edat (Espino Guarch et al., 2018). Així i tot, el seu mecanisme de transport complet i en detall encara resta desconegut, atès que la gran majoria d’estructures resoltes fins a l’actualitat es troben en el mateix estat obert al citosol, amb la única excepció de LAT1 unit a inhibidors resolt tancat pel citosol (Yan et al., 2021). Amb la finalitat de revelar el mecanisme de transport dels LATs ens proposem emprar com a model l’intercanviador obligat bacterià d’alanina-serina-cisteïna (BasC). Aquest intercanviador ja ha estat prèviament caracteritzat funcionalment (Bartoccioni et al., 2019) i la seva estructura es va resoldre en complex amb el nanocòs específic 74 (Errasti-Murugarren et al., 2019). Els 29 nanocossos han sigut caracteritzats pels seus paràmetres d’unió, el seu costat d’interacció amb BasC i l’efecte conformacional que efectuen sobre BasC per assajos de transferència d’energia de ressonància fluorescent per molècules individuals (smFRET) sobre el TM1a de la porta citosòlica de BasC. Els estudis de smFRET ens han permès estudiar la dinàmica de tancament de la porta citosòlica induïda per l’adició de substrats, L-Ala i L-Ser. A més, s’ha comprovat que els nanocossos poden esdevenir una eina molt útil a l’hora de bloquejar certs moviments de la proteïna i, eventualment, bloquejar el transportador en un estat conformacional diferent del ja resolt, seleccionant els nanocossos prometedors per resoldre l’estructura de BasC en complex amb ells per tècniques estructurals com la cristal·lografia de raigs X o la crio-microscopia electrònica. Finalment, aquests efectes de tancament de la porta citosòlica induïts pels substrats van ser assajats sobre la variant relacionada amb la malaltia d’intolerància lisinúrica proteica de BasC (K154A). Aquesta variant ha resultat ser incapaç de tancar la porta citosòlica, esdevenint la primera evidència experimental sobre el rol clau d’aquest residu, K154, en el mecanisme de transport dels LATs, més concretament el tancament de la porta citosòlica. Al cap i a la fi, aquest projecte pretén presentar i posar a punt una tècnica que pot ser d’alta utilitat per estudiar el mecanisme de transport de proteïnes de membranes, així com els funcionament aberrants de les variants relacionades amb les malalties.