Linking function to global and local dynamics in an elevator-type transporter

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt_Ph. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Transporters are integral membrane proteins that move solutes across lipid bilayers. They undergo concerted conformational changes, allowing alternate exposure of their substrate-binding sites to external and internal solutions (1). In each of these so-called outward- and inward-facing states (OFS and IFS, respectively), further isomerizations accompany substrate binding and release. Transport efficiency depends on the rates of these rearrangements, but linking function and structural dynamics has presented methodological challenges. Single-molecule Forster resonance energy transfer (smFRET)-based total internal reflection fluorescence (TIRF) microscopy (2–4) has been used to monitor the dynamics of the OFS to IFS transitions (5–8) and single-transporter activity (9) in the elevator-type transporter Glt_Ph and other transporters (10–18). Hydrogen–deuterium exchange followed by mass spectrometry (HDX-MS) has been used to pinpoint local changes in structural dynamics in diverse biological systems (19–21). Here, we combine these approaches to link changes in local protein dynamics to the larger-scale conformational transitions and substrate transport in wild-type (WT) and gain-of-function mutants of Glt_Ph.Glt_Ph is an extensively studied archaeal aspartate transporter that is homologous to human excitatory amino acid transporters (EAATs). Structures of Glt_Ph (7, 22–30), and archaeal and mammalian homologs (31–37), show that the transporters assemble into homotrimers via scaffold domains. Each protomer features a mobile transport domain that binds l-Aspartate (l-Asp) and three Na⁺ ions (22, 23, 28, 31, 38) and symports the solutes by an elevator mechanism, moving ∼15 Å across the membrane from an OFS to an IFS (6, 8, 23, 24, 39). During the elevator transitions, two structurally symmetric helical hairpins (HPs) 1 and 2 form the cores of the domain interfaces in the OFS and IFS, respectively (SI Appendix, Fig. S1A) (23, 24, 40). Despite symmetry, they do not have the same function. HP1 is mostly rigid, while HP2 is a conformationally plastic “master regulator” of the transporter, gating substrate in the OFS and IFS and contributing to setting the elevator transition rates (5, 23, 24, 27, 29, 36, 41–47).In this study, we use three previously characterized mutants of Glt_Ph to pinpoint the rate-limiting steps of the transport cycle and probe the protein dynamic properties that correlate with increased transport rates. A K290A mutation at the base of HP1 disrupts a salt bridge with the scaffold domain in the OFS and dramatically increases the elevator dynamics (5, 6). A triple-mutant Y204L/A345V/V366A displays a more modest increase in elevator dynamics and substantially diminished l-Asp affinity (5). Finally, a Y204L/K290A/A345V/V366A mutant combines these substitutions and their effects (5). We compared our previously obtained smFRET data on the elevator dynamics of the WT transporter and the mutants (5) to single-transporter uptake measurements. For WT Glt_Ph, these dynamics and transport measurements established transporter subpopulations that move (5, 6) and work (9) with rates differing by orders of magnitude, with slow transporters dominating the ensemble. We now show that only mutations that both reduce the population of the slow-moving transporters and weaken substrate affinity, such as Y204L/A345V/V366A, reduce the population of the slow-working transporters and confer overall gain-of-function properties. The slow-working population comprises transporters with rare elevator transitions or slow substrate release. We then used HDX-MS to explore how the Y204L/A345V/V366A mutant differed from the WT protein. We found that the mutations decreased the stability of the secondary structure around the substrate-binding site, suggesting that the increased local dynamics underlie reduced kinetic heterogeneity within the mutant transporter ensemble.