iiinova — where your digital self keeps evolving

Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

14/14 If you’ve made it this far, I really appreciate it (and also go touch grass). My lab is pushing this work in new directions and incorporating more fun, interdisciplinary techniques to understand transporter structure-function mechanism in unique ways. If this sounds interesting, reach out!

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

11/14 This means ion coupling isn’t solely dictated by ion-binding residues, but from allosterically regulated packing - a structural “clutch” linking ion/substrate binding. We think this applies to ion-coupled transport and beyond - small, distant mutations can flip fundamental energy landscapes.

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

10/14 What changed? Our evolutionary analysis gave us an elegant answer. A central ‘coupling’ helix is responsible for cooperative ion/substrate binding, and two changes at the start and end of this helix can turn sodium coupling on/off. These residues control how rigidly the helices pack together.

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

9/14 Cryo-EM with precise sample conditions and processing gave us the clue: the intermediate ancestor could spontaneously access the high-affinity substrate-binding state required for transport, which sodium-coupled transporters can only reach with sodium.

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

8/14 To our surprise, an ‘intermediate’ ancestor during the transition from sodium to proton coupling still had sodium binding sites, could bind sodium, but no longer needed sodium energy for substrate binding and transport - an ion-independent, ‘uncoupled’ transporter. How?

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

7/14 Our ancestral membrane proteins had terrible yields, making purification/characterization a pain. We reinvented all the lab pipelines to make this project work. I like to call this the protein purification of Theseus. If you replace every step of a protocol, is it the same protocol? 🤔

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

6/14 Since loops and tails are poorly reconstructed, we had to stitch in some loops/tails from existing proteins for expression constructs, which @olgabiophys.bsky.social affectionately called “Frankensteins”…which of course makes me Dr. Frankenstein.

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

5/14 We borrowed a page from their playbook, using ancestral protein reconstruction. Essentially, we use phylogenetics to approximate how evolution might have occurred, and generate inferred ‘ancestral’ transporter sequences spanning this functional transition. Great review: doi.org/10.1146/annu...

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

4/14 For years, we did sequence alignments and mutated residues that looked interesting, to no avail. These processes might be too complex/allosteric - inspired by beautiful work from @joethorntonlab.bsky.social, we thought understanding the evolutionary process could be an avenue to untangle this.

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

3/14 Brain glutamate transporters use sodium to recycle neurotransmitters. Close relatives in bacteria also use sodium…but others use protons. We wanted to understand how transporters made this switch. What changes in sequence made this possible? And what molecular features enforce ion coupling?

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Krishna Reddy @krishnareddy.bsky.social · 12/09/2025

2/14 Secondary active transporters perform ‘concentrative’ transport, moving substrates against their concentration gradients by harnessing energy from ion gradients. Organisms have evolved their transporters based on their environment – i.e., halophiles often have sodium-driven transporters.

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