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Prof. Cristina Paulino

Department of Structural Biology, University of Groningen

From bacteria to humans: deciphering the mechanism of membrane proteins by cryoEM at 200 kV

"I will present two projects that highlight our focus in understanding the structure-function relationship of membrane transporters. The first story will focus on the rare bacterial K+ uptake system KdpFABC, which is a unique chimera composed of a primary-active transporter and a channel-like domain. While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K+ has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. We were able to determine two additional structures of the 157 kDa, asymmetric KdpFABC complex by cryo-EM in an E1 and E2 state, at 3.7 Å and 4.0 Å resolution, respectively. Unexpectedly, the new structures suggest a so far unprecedent transport mechanism through two half-channels along KdpA and KdpB, uniting the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K+ channels. This way, KdpFABC functions as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms.

In the second half I will present our recent work on the human neutral amino acid transporter ASCT2 (SLC1A family). As the main transporter of glutamine into the cells, ASCT2 is upregulated in a large variety of different cancer types and a potential anti-cancer drug target. However, to date our understanding of the SLC1 transporters has been limited to bacterial or heavily modified human homologues. We were able to determine the first ASCT2 structure and resolve a long lasting key mechanistical question relevant for all SLC1A members. We demonstrate that during translocation the same structural element acts as a gate for substrate-binding and -release on both sites of the membrane. We conclude thet SLC1A transporters operate by a one-gate, and not as intially assumed by a two-gate, elevator mechanism. Further, we could identify several potential allosteric binding sites that might guide the design of lipidic-inhibitors for anticancer therapy.

Notably, these studies among others performed in the lab show the potential of 200 kV TEM machines for the structure determination of small and challenging membrane proteins."