Biology subjects

Sauer, D. B.

Publications and source records attributed to Sauer, D. B..

8 recordsLinked to original sources

bioRxiv2026

Structural Basis for Inhibition of Human Anion Transporter SLC26A6/PAT-1

PAT-1, encoded by the gene SLC26A6, is an exchanger of small organic and inorganic anions including oxalate and chloride in the kidney and the intestines. PAT-1 dysfunction is implicated in nephrolithiasis and hyperoxaluria, and its modulation is a potential therapeutic strategy to treat cystic fibrosis. PAT-1 has two small molecule inhibitors: DIDS, a non-specific anion exchanger inhibitor; and PAT1inh-B01 which is highly specific to PAT-1. In this study, we report high-resolution cryo-electron microscopy structures of the human PAT-1 homodimer in complex with these inhibitors to reveal their different inhibition mechanisms. These structures also uncover the complex interactions between the transmembrane, STAS and N-terminal domains, with implications on its ion exchange function. Hence, this study provides mechanistic insight into PAT-1 molecular function and inhibition, and contributes to our understanding of SLC26 transport mechanisms.

biochemistry↗

bioRxiv2026

Structures of the human sodium-citrate cotransporter NaCT with and without substrates

The human sodium-citrate cotransporter NaCT imports various tri- and dicarboxylates into the cell as TCA cycle intermediates. This substrate uptake process is driven by an inward sodium gradient. The protein is a member of the Divalent Anion-Sodium Symporter (DASS) family. Whereas extensive biochemical and structural studies have been carried out for NaCT, how the substrate binding and translocation is coupled to the sodium gradient remains unclear. Here using single particle cryo-electron microscopy, we determined the structures of the human NaCT protein in three states: sodium-free, in the presence of sodium, and sodium- and substrate-bound. These structures suggest a simultaneous binding mechanism for sodium-substrate coupling, distinct from the sequential binding, conformational selection mechanism previously observed for the bacterial DASS protein VcINDY.

biophysics↗

bioRxiv2024

Structures of the human adult muscle-type nicotinic receptor in resting and desensitised states

Muscle-type nicotinic acetylcholine receptor (AChR) is the key signalling molecule in neuromuscular junctions. Here we present the structures of full-length human adult receptors in complex with Fab35 in -bungarotoxin (BuTx)-bound resting states, and in acetylcholine (ACh)-bound desensitised states. In addition to identifying the conformational changes during recovery from desensitisation, we also used electrophysiology to probe the effects of eight previously unstudied AChR genetic variants found in congenital myasthenic syndrome (CMS) patients, revealing they cause either slow- or fast-channel CMS characterised by prolonged or abbreviated ion channel bursts. The combined kinetic and structural data offer a better understanding of both AChR state transition and the pathogenic mechanisms of disease variants.

neuroscience↗

bioRxiv2024

Metabolic mapping of the human solute carrier superfamily

Solute carrier (SLC) transporters govern most of the chemical exchange across cellular membranes and are integral to metabolic regulation, which in turn is linked to cellular function and identity. Despite their key role, individual functions of the members of the SLC superfamily were not evaluated systematically. We determined the metabolic and transcriptional profiles upon SLC overexpression in knock-out or wild-type isogenic cell backgrounds. Targeted metabolomics provided a fingerprint of 189 intracellular metabolites, while transcriptomics offered insights into cellular programs modulated by SLC expression. Beyond the metabolic profiles of 102 SLCs directly related to their known substrates, we also identified putative substrates or metabolic pathway connections for 71 SLCs without previously annotated bona fide substrates, including SLC45A4 as a new polyamine transporter. By comparing the molecular profiles, we identified functionally related SLC groups, including some with distinct impacts on osmolyte balancing and glycosylation. The assessment of functionally related human genes presented here may serve as a blueprint for other systematic studies of human gene function and supports future investigations into the functional roles of SLCs.

systems biology↗

bioRxiv2024

Di-Gluebodies as covalently-rigidified, modular protein assemblies enable simultaneous determination of high-resolution, low-size, cryo-EM structures

Cryo-EM has become a routine structural biology method, yet elucidation of small proteins (<100 kDa) and increasing throughput remain challenging. Here, we describe covalently-dimerized engineered nanobodies, Di-Gluebodies, as novel and modular imaging scaffolds to address the challenges. They can simultaneously display two copies of the same protein or two distinct proteins, providing sufficient rigidity required by cryo-EM. We validate this method with multiple soluble and membrane targets, demonstrating that this provides a flexible and scalable platform for expanded protein structure determination.

synthetic biology↗

bioRxiv2023

Structural basis of the mechanism and inhibition of a human ceramide synthase

Ceramides are bioactive sphingolipids that play pivotal roles in regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by a family of six ceramide synthase enzymes (CerS), each with distinct specificity for the acyl-CoA substrate. Importantly, the acyl chain length plays a key role in determining the physiological function of ceramides, as well as their role in metabolic disease. Ceramide with an acyl chain length of 16 carbons (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease, and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here, we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, together with biochemical characterization using intact protein and small molecule mass spectrometry, reveal that CerS catalysis proceeds via a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, providing new insights into its inhibition of CerS. These results provide a framework for understanding the mechanisms of CerS function, selectivity, and inhibition, and open new directions for future drug discovery targeting the ceramide and sphingolipid pathways.

biochemistry↗

bioRxiv2023

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes which link substrate release and opening of the cytoplasmic gate, and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

biophysics↗

bioRxiv2022

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters. Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, "elevator-type" motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.

biophysics↗