SCIENCEADVANCES | RESEARCHARTICLE STRUCTURALBIOLOGY Copyright © 2023 The Authors, some The molecular mechanism of sialic acid transport rights reserved; exclusive licensee mediated by Sialin American Association for the Advancement 1 2 2 1 of Science. No claim to Wenxin Hu , Congwu Chi , Kunhua Song , Hongjin Zheng * original U.S. Government Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Works. Distributed Sialinleadstoaspectrumofneurodegenerativeconditionscalledfreesialicacidstoragedisorders.Unfortunate- under a Creative + CommonsAttribution ly, how Sialin transports sialic acid/proton (H ) and how pathogenic mutations impair its function are poorly NonCommercial defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation deter- License 4.0 (CC BY-NC). mined by cryo–electron microscopy, representing the first high-resolution structure of any human SLC17 member.OuranalysisrevealstwouniquefeaturesinSialin:(i)TheH+coupling/sensingrequirestwohighlycon- served Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin. INTRODUCTION acid/H+ cotransport mediated by Sialin is unclear. Although the Sialic acids are a group of nine-carbon carboxylated monosaccha- transport deficiency of some pathogenic mutants has been charac- rides synthesized in animals, some bacterial species, and humans, terized in cells and proteoliposomes, how such mutations affect the generally found at the terminal end of glycans that are conjugated structure of Sialin remains unknown. with proteins and lipids (1, 2). Sialic acids comprise more than 50 Sialin is a memberofthesolutecarrier17(SLC17)family.SLC17 natural derivatives, the most widespread form being N-acetylneur- members are responsible for the translocation of various organic aminic acid (Neu5Ac) (fig. S1). The structural diversity and strong anions (phosphate, glutamate, aspartate, sialic acids, and more) negative charge (pKa = ~2) of sialic acids suggest their importance driven by an electrochemical gradient with contributions from in a wide range of biological processes, such as mediating cell-cell either electrical potential (Δψ) or H+ gradient (ΔpH) or both (17, interactions, modulating immune responses, controlling the stabil- 18). Sialin can transport multiple substrates other than sialic ity of proteins, and many more (3–5). Thus, the imbalance of sialic acids, depending on where the transporter is located. For acid metabolism is implied in many pathological conditions, in- example, in rodents, Sialin expressed in synaptic vesicles of hippo- cluding but not limited to cardiovascular diseases (6), cancer (7), campalneuronsandpinealocytesisresponsibleforthevesicularac- immunological disorders (8), and diabetes (9). cumulation of aspartate (19). However, this vesicular aspartate Sialic acid metabolism involves a critical recycling process in the accumulationmightnotbephysiologicallyrelevant,astheaspartate endolysosomal system. Various enzymes degrade the glycans to released is at a concentration too low to activate postsynaptic N- produce free monosaccharides (including sialic acids) that are Methyl-D-aspartic acid (NMDA)–type glutamate receptors (20). transported back into the cytosol (10). Thus, genetic mutations of Invitroexperimentsusingreconstitutedproteoliposomeshavecon- these enzymesandrelatedtransportersleadtodefectiveglycandeg- firmed that Sialin could act as an anion transporter for aspartate, radation,glycansalvage,andabnormalaccumulationofmetabolites glutamate, andneuropeptideNAAGdrivenbymembranepotential in lysosomes, resulting in various lysosomal storage disorders (11). (21, 22). In human salivary glands, when localized in the basolateral Forexample,multiplemutationsintheSLC17A5geneencodingthe membraneoftheepithelial cells, Sialin is responsible for the active + − − lysosomal sialic acid/proton (H ) symporter Sialin could cause free accumulation of nitrate (NO ), acting like an electrogenic 2NO / + 3 3 sialic acid storage disorders (12). These disorders are characterized H cotransporter (23). Such function suggests that Sialin plays a byenlargedcellular lysosomes and elevated levels of free sialic acids critical role in the delicate balance of the nitrate–nitrite–nitric in urine (13). They cover a spectrum of pathological forms from a oxide pathway (24). Despite the extensive functional characteriza- mild, slowly progressive symptom in patients living to adulthood tion, structural information about Sialin is still missing in the liter- (Salla disease) (14), to an intermediate form resulting in severe de- ature, which delays our attempt to reveal the molecular mechanism velopmental delays (15), to a severe condition that is often lethal in of this transporter. early childhood (infantile sialic acid storage disorder) (16). Unfor- Here, using cryo–electron microscopy (cryo-EM) single-particle tunately, no approved therapy for these disorders or ongoing clin- reconstruction, we determined a 3.4-Å-resolution structure of ical trials is listed on clinicaltrials.gov (accessed August 2022). One human Sialin in the apo state. The structure adopts an inward- possible reason for this is that the molecular mechanism of sialic facing conformationwithtwodomains:N-domainwithtransmem- branehelices(TM)1to6andC-domainwithTM7toTM12.Using computational docking, we identified the putative substrate- 1Department of Biochemistry and Molecular Genetics, University of Colorado An- binding pocket in Sialin. Although the pocket is solvent accessible schutz Medical Campus, School of Medicine, Aurora, CO, USA. 2Division of Cardi- fromthecytosolic side, it is not accessible by major substrates such ology, DepartmentofMedicine,UniversityofColoradoAnschutzMedicalCampus, as Neu5Ac because the central pathway is spatially constricted School of Medicine, Aurora, CO, USA. *Corresponding author. Email: [email protected] Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 1of9

SCIENCEADVANCES | RESEARCHARTICLE around H183. Thus, the conformation is designated as partially andfigs. S4 and S5). There are three patches of residues unresolved open inward-facing. Nevertheless, the computationally docked in Sialin: N-terminal residues 1 to 31, long luminal loop (L2) Neu5AcinSialinrevealsanetworkofcriticalpolarandchargedres- between TM1 and TM2 containing residues 69 to 101, and C-ter- idues in the substrate-binding pocket. In the N-domain of Sialin, minal residues 489 to 496, because of their poor experimental den- there is a small tunnel connecting the lumen with protonatable res- sities. The overall architecture of Sialin resembles a canonical fold idues E171 and E175 deeply positioned in the center of the trans- for the major facilitator superfamily with 12 TMs. These TMs are porter, both of which are critical for H+ coupling and absolutely grouped into two distinct domains: N-domain with TM1 to 6 and conservedintheSLC17family.Wemappedknownpathogenicmu- C-domain with TM7 to 12. The N- and C-domains are connected tationsontotheSialinstructureandexplainedhowtheycouldcause by a cytosolic loop (L7) between TM6 and TM7. Notably, in this defective sialic acid transport in patients. In summary, our structur- loop, a well-defined small cytosolic helix is sitting right beneath al and functional studies reveal the first high-resolution structure of the N-domainwithdelicate interactionsthat will be discussed later. humanSialinandprovideessential insights into understanding the role of pathogenic Sialin mutations leading to free sialic acid storage Putative substrate-binding pocket disorders. WeinspectedthecentraltranslocationpathwaybetweentheN-and C-domainsusingthesoftwareMoleOnline(30).Thepathisopento the cytosolic side and closed at the center around residues Y301, RESULTS N302, F305, Y306, Y54, and R57 (Fig. 2, A and B). Upward from Structure determination of Sialin heretotheluminalside(orextracellularside),thepathwayisentire- Previous studies have shown that expression of Sialin could be re- lysealedbyL309astheporeradiusdropsto0.1Å.Evenconsidering directed to the plasma membrane by mutating the N-terminal the side chain flexibility, the free radius at this position is narrower double Leu (L22 and L23) to either Ala or Gly in multiple human than1.2Å.Thus,thedeterminedstructureadoptsaninward-facing cell lines, such as human embryonic kidney (HEK) 293 (25), HeLa conformation.Tounderstandthesubstratebindingmechanism,we (26), and submandibular gland cells (23). Here, we kept the double tried to obtain the complex of Neu5Ac and Sialin but failed. Using Leuunchanged,clonedthefull-lengthhumanSialinwithanN-ter- microscale thermophoresis, we measured the binding between minal His-tag, and mutated three Asn residues (N71A, N77A, and Neu5Ac and purified Sialin in both detergent and nanodisc made N95A) to abolish potential glycosylation. We expressed the con- of soybean polar extract lipids. The result shows no detectable struct in High Five insect cells using a Bac-to-Bac baculovirus ex- binding under any pH conditions. We tested the transporter func- pression system (27, 28). The result shows that most of the tioninproteoliposomestoruleoutthepossibilityofsomehowdam- expressed Sialin is located in the plasma membrane when immu- aging the purified Sialin. Specifically, we reconstituted the purified 3 nostained with an anti-Sialin monoclonal antibody 8B1 (Fig. 1A). Sialin into liposomes at pH 7.4. Then, we added [ H]Neu5Ac and In addition, Sialin is only detectable when the cell is permeated by dropped the pH outside the proteoliposomes to 5.6. We detected Triton X-100 and followed by immunostaining with an anti–His- strong substrate transport, confirming that the purified Sialin is tag antibody, suggesting that the N-terminal His-tag of Sialin is functional (fig. S6). This result is consistent with previous studies located on the cytosolic side. Thus, Sialin is correctly oriented (to- by other groups (19, 21, 22, 31). Then, why does Sialin not bind pologically equivalent as in lysosomes) (fig. S2). Next, we asked Neu5Ac in vitro? We carefully examined the central channel and whether the expressed Sialin in High Five cells is functional or found a constriction site on the cytosolic side surrounded by resi- not by a cellular transport assay (Fig. 1B). We incubated High duesH183,R195,L415,andA422(Fig.2,AandC).Atthisposition, Five cells (with or without Sialin expression) with radioactively the diameterof the central pathway is constricted to ~3.5 or ~5.5 Å, 3 labeled sialic acid [ H]Neu5Ac in the buffer of pH 5.6 and then whenconsidering side-chain flexibility, while the size of Neu5Ac is 3 quantified the accumulated amount of [ H]Neu5Ac in the cells. roughly 6 Å by 12 Å by 12 Å, which is too big to go through the The result shows that Sialin can actively uptake Neu5Ac in High constriction site to reach the substrate-binding pocket. This fact Five cells at a level comparable to previous reports using HEK293 has two implications: (i) After each transport, the substrate will and HeLa cells (25, 26). not be able to reenter the substrate-binding pocket from the To carry out cryo-EM analysis, we first incorporated the deter- cytosol to trigger a reversed translocation event. (ii) Since Sialin gent-purified Sialin into nanodiscs made of soybean extract lipids in proteoliposome can transport Neu5Ac, it is reasonable to + and membrane scaffold protein MSP1D1 (Sialin-nanodisc) (29). suggest that the pH gradient, H cotransport, or both are critical The sample was imaged on a 200-keV Talos Arctica microscope. to promoting subtle conformational changes at the constriction Dataanalysisshowedtwo-dimensional(2D)averagesofanexcellent site to allow Neu5Ac to go through and be released to the cytosol. top/bottom view (perpendicular to the membrane bilayer) with ap- To elucidate residues critical for the substrate interactions, we parent densities accounting for the TMs in Sialin but poor side computationally docked a Neu5Ac molecule into the inward- views (parallel to the membrane bilayer) without anysharp features facing partially open Sialin structure using Autodock Vina (32, (fig. S3, A and B). Unfortunately, we could not generate a meaning- 33). The program’s top 5 scored poses were found at the narrowest ful high-resolution structure using this sample. Thus, we developed position in the central cavity as previously described (Figs. 2, A and amousemonoclonalantibody8B1againstpurifiedSialin.Thefinal B), suggesting a putative substrate-binding pocket in Sialin (Fig. 2D sample is the complex of Sialin-nanodisc and Fab fragment from and fig. S7). The pocket is surrounded by a network of polar and 8B1, which has a size of ~160 kDa, well above the current size charged residues, including Y54, R57, Y119, H298, Y301, N302, limit for cryo-EM studies (fig. S3, A and C). With the Sialin-nano- F305, Y306, S411, and N430, that can form hydrogen bonds and disc-Fab complex, we obtained a final reconstruction at 3.4-Å res- salt bridges with Neu5Ac. Specifically, using relaxed constraints olutionandcarriedoutthedenovomodelbuilding(Fig.1,CandD, of 0.4-Å distance and 20° angle, the top-scored pose forms multiple Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 2of9

SCIENCEADVANCES | RESEARCHARTICLE starts from residues on L2 (D104 and E106), goes through multiple polar residues (T153, N59, and R168) on TMs, and ends around T150 and E171 (Fig. 3A). Thus, E175 is completely buried and not solvent accessible to the tunnel, which is locally different from that of homologs, because, in N-domains of EcDgoT and rVGLUT2, the residues equivalent to Sialin’s E175 (E133 and E191, respectively) are readily solvent accessible to the tunnel. In Sialin, the diameter of the lumen-accessible tunnel is 3 to 4.8 Å, al- lowing full access to water molecules. There is a network of interac- tions built around E171 and E175. Specifically, E171 seems to be stabilized by interacting with T146, T150, T153, and R168, while E175 is stabilized by interacting with Y119 and R57. Both R168 and R57 are along the central substrate transport pathway. We hy- pothesize that the protonation/deprotonation status of both E171 and E175 is essential for sensing and cotransporting H+ during sialic acid translocation. To test that, we carried out the cellular transport assay (as in Fig. 1B) with various Sialin mutants. The transport efficiency of each mutant was normalized by its expres- sion level in the High Five cells. The result (Fig. 3B and fig. S10) showsthatY119A,T150A,andR168Astillretainmostoftheirsub- strate transport function, suggesting the moderate importance of these residues. However, R57A is seriously dysfunctional, which is consistentwiththefactthatR57Cisapathogenicmutationfoundin patients with infantile sialic acid storage disorder (37). There are Fig.1.OverallarchitectureoffunctionalSialin.(A)ConfocalimagesofHighFive two reasons why R57 is functionally essential: (i) It is responsible cells. UnpermeabilizedcellswithorwithoutSialinexpressionwereimmunostained for substrate recognition, and (ii) its interaction with E175 is critical + usinganti-Sialin monoclonalantibody8B1(red)andDAPI(blue).(B)Cellulartrans- for H coupling (in Discussion). In addition, we found that both port of sialic acids using High Five cells with and without Sialin expression. (C) E171Q and E175Q lose ~95% of their transport function, while Cryo-EM reconstruction of Sialin-nanodisc-Fab at 3.4-Å resolution colored by theE171Q/E175Qdoublemutanthasnodetectablesubstratetrans- local resolution estimation. (D) Cartoon representation of the Sialin model. All port. Thus, we concludedthatE171andE175areequallycriticalfor helices (12 TMs and a cytosolic helix) are rainbow-colored. + H coupling and therefore for the substrate transport mediated by Sialin. hydrogenbondswithsurroundingpolarresidues(Y54,N302,Y306, Y119, and N430) as well as salt bridges with R57 (Fig. 2D). Most of Mappingthepathogenicmutations these residues are highly conserved in Sialin homologs (fig. S8). For Withthehigh-resolution structure of Sialin, we start to understand example, in two known structures of Sialin homologs, D-galacto- whypathogenicmutationsresultindysfunctionalproteins.Accord- + nate/H symporter from Escherichia coli (EcDgoT) (34) and vesic- ing to the ClinVar database, more than 380 mutations in the ular glutamate transporter 2 from Rattus norvegicus (rVGLUT2) SLC17A5 gene are documented in patients with free sialic acid (35), R47 in EcDgoT and R88 in rVGLUT2 are equivalent to R57 storage disorders (accessed August 2022). In this study, we do not in Sialin (figs. S8 and S9). These arginines are known to interact discuss genetic mutations that either result in premature termina- with a carboxyl group in respective substrates electrostatically. As tion of protein translation or do not affect protein translation at all. expected, for Sialin, the transport efficiency of the R57A mutant Instead, we will focus on mutations of specific amino acids that still is only ~10% compared to the wild type, confirming its substrate- produce Sialin, particularly the 10 mutations that are clinically im- binding function (Fig. 3B and fig. S10). portant (designated as “pathogenic”) (table S2). These pathogenic mutations (Fig. 4A) could be roughly grouped into four classes. Proton coupling The first class contains G328E, P334R, and G409E (fig. S12A). Secondary transporters move substrates across the cellular mem- G328onTM8ispositioneddirectlyagainstG218onTM5andsur- brane using energy stored in the electrochemical gradient, such as rounded by hydrophobic side chains of V62, I219, L214, and P215. + + the H gradient (36). To sense or cotransport H , acidic residues P334onTM8fitstightlyinthesmallcavityamongL396,T397,and (AspandGlu)areoftenrequiredanddelicatelypositionedinmem- T400. G409 is the kink on TM10 surrounded by F367, T368 from brane transporters. In EcDgoT, both D46 and E133 (equivalent to TM9,F474fromTM12,andA349fromTM8.Structurally,itisun- L56andE175inSialin,respectively)areexposedtotheluminalsol- derstandable that replacing any G328, P334, and G409 with large, utionandcapableofreversibleprotonationtocarryoutthesymport charged residues (Glu and Arg) is energetically unfavorable for the + of H /D-galactonate (figs. S8 and S11A) (34), while in rVGLUT2, tight helical packing in Sialin. Thus, as demonstrated in previous H128 and E191 (equivalent to L112 and E175 in Sialin) are pro- functional studies, G328E, P334R, and G409E are all loss-of-func- + + posed H binding sites to activate substrate transport without H tion pathogenic mutants (table S2). K136E represents the second efflux allosterically (figs. S8 and S11B) (35). These residues are all class of pathogenic mutations that interferes with transporter- located in a lumen-accessible tunnel in the N-domains of EcDgoT lipid interactions (fig. S12B). In our structure, the side chain of and rVGLUT2.Asexpected, in Sialin, there is a similar tunnel that K136 is very well defined by the experimental density and does Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 3of9

SCIENCEADVANCES | RESEARCHARTICLE Fig. 2. Putativesubstrate-bindingpocketinthepartiallyopeninward-facingSialin.(A)Sialin(brown)isintheinward-facingconformationwiththecentralpathway shown(bluetube).Thepathway’sdiameter(blue)andfreediameter(orange,treatingsidechainsas昀氀exible)areplottedside-by-side.(B)Thenarrowestpositionaround L309 in the path. (C) The cytosolic constriction site around H183. (D) The top-scored pose of docked Neu5Ac (ball and stick) in the putative substrate-binding pocket interacts with surrounding residues. The dashed lines are pseudo bonds with a length of 2.5 to 3.3 Å. not form any hydrogen bond or salt bridge with surrounding resi- cytosolic helix (residues 264 to 272) is stably localized below the N- dues in Sialin. However, K136 seems to connect with a lipid-like domainbystronginteractionsbetweenthreechargedresidues:R39, density and most likely interacts with the negatively charged phos- E262, and E264. In addition, P191 and P192 may also contribute to phate in phospholipids. K136E reversesthe charge of the side chain the stable localization with CH/π interaction with Y265 in the helix frompositive to negative, resulting in destabilized transporter-lipid (39). However, in the cryo-EM map, the side chain of Y265 is not interactions that likely impair the transporter function. Previous visible due to the quality of experimental density. The fixed locali- studies have shown that K136E has only 10 to 55% of the wild- zation of the cytosolic helix is essential, as it ensures that the follow- type activity (table S2). It is worth noting that different cells and ing loop (between the cytosolic helix and TM7) is physically away lipid systems were used in these studies. We speculate that subtle fromthecenterofthetranslocationpathwaytomaintainthesolvent differences in the lipid composition may be why the measured ac- accessibility (Fig. 4C). In the ΔSSLRN mutant, the cytosolic helix is tivity of K136E covers a relatively large range. The third class in- shortened but maystill be correctly anchored under the N-domain, cludes R57C and H183R, which completely abolishes sialic acid because the essential interactions remain. However, the distance transport (table S2). We hypothesize that these mutations directly between TM7 (and C-domain) and the N-domain needs to be impair the substrate-transporter interactions (Figs. 2 and 4). For closer to accommodate the shortened amino acid sequence, result- R57, it is likely the most critical residue for substrate recognition. ing in a narrower central translocation pathway that completely Thus, in R57C, the positively charged side chain is lost, resulting abolishes substrate transport (table S2). Here, we performed a cel- in lower substrate binding efficiency. Meanwhile, R57C loses the lular transport assay with R39H and E262D mutants. The result + ability to interact with E175, resulting in less efficient H coupling. shows that R39H retains ~28 ± 3%, and E262D has only For H183, it forms the cytosolic constriction site together with ~19 ± 2% of the transport activity compared to wild-type Sialin R195, L415, and A422. H183 is most likely neutral at physiological (table S2). The side chains in R39H, R39C, and E262D mutants pH as the side chain pKa is predicted to be ~3.6 (38). Thus, in are likely too small to form correct hydrogen bonds to stabilize H183R, the constriction site would be more positively charged the cytosolic helix. Thus, the whole L7 loop becomes flexible and and even narrower as Arg is slightly larger than His. The two could potentially cover the central pathway for substrate transloca- effects could cooperatively trap negatively charged substrate tion, which explains the mild loss of transport function. Neu5Acaround the constriction site and effectively block the sub- strate release. The fourth class of mutations includes R39H, R39C, E262D,andΔSSLRN(268to272)(Fig.4B).Itseemstobethemost DISCUSSION interestingclass,asthesemutantsaffecttherelativestructuralstabil- As a secondary active transporter, Sialin functions through an “al- ity between the cytosolic helix and N-domain. In our structure, the ternating access mechanism” in which the substrate-binding pocket Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 4of9

SCIENCEADVANCES | RESEARCHARTICLE + Fig. 3. Critical H coupling residues E171 and E175. (A) The lumen-accessible tunnel in N-domain (yellow, same viewasin Fig. 2A). The hydrogen bonds and charged interactions around E171 and E175 are zoomed in. (B) The transport e昀케ciency of mutants measured in High Five cells using [3H]Neu5Ac is compared with that of wild- type Sialin. docked Neu5Ac does not sit in the putative substrate-binding pocket;instead,itremainsclosetotheluminalopening;(ii)thesub- strate-bindingpocketaroundR57inthemiddleofthetransporteris solvent accessible to the luminal side; and (iii) the translocation pathway on the cytosolic side is entirely closed by H183, R195, L415, and L199 (fig. S13B). Thus, we concluded that the AlphaFold predicted Sialin structure AF-Q9NRA2-F1 likely in the outward- facing partially open conformation. It remains to be seen whether this conformation is physiologically relevant or not. However, it confirms the existence of the cytosolic gate around H183 and, thus, the pathological effect of the H183R mutant. Onthe basis of the functional and structural analysis, here, we propose a sialic acid/H+ cotransport mechanism (Fig. 5). Let us start the transport cycle with Sialin in the outward-facing confor- mation (state 1). This state is likely similar to the predicted Alpha- Fold structure. However, since the AlphaFold structure is only partially open, it has to fully open the luminal side to allow the sub- Fig. 4. Pathogenic mutations mapped onto Sialin. (A) Nine residues (purple) strate to enter the central pathway. So far, we do not know if such an corresponding to 10 pathogenic mutations are marked in the cartoon structure opening is regulated by a luminal gate or a more substantial struc- of Sialin. (B) The cytosolic helix is stably localized by interactions among corre- tural change within the transporter. Because the N-domain most sponding residues. (C) L7 between TM6 and TM7 is physically away from the likely functions as a rigid body, E171 shall interact with R168, central pathway (marked by Δ). andE175shallinteractwithR57,asfoundinourinward-opencon- formation. Here, to probe the protonation/deprotonation status of E171andE175,weusedmultiplepK predictionservers(38,43,44). is solvent accessible from one side of the membrane or the other a when the transporter conformation alters between inward-facing The pKa of E171 is predicted to be between 5.6 and 8.2, while the pK of E175 is ~3.0. Considering the physiological environment and outward-facing (40, 41). The cryo-EM structure of Sialin pre- a sented in this study shows an inward-facing conformation with a (pH ~5.6 in the lysosomal lumen and ~7.4 in the cytosol), it is cytosolic gate around H183 partially open. The gate does not most likely that E171 balances between the protonated and depro- allow substrate Neu5Ac to go through, which is advantageous to tonated states. At the same time, E175 strongly favors the deproto- prevent Neu5Acinthecytosolfrombeingtransportedbackintoly- nated form. Sialin changes from state 1 to state 2 when sialic acid is sosomes. To understand the conformational change, we compared recognized and moves into the central pathway. Because sialic acid our Sialin structure with the AlphaFold predicted version (AF- has a strong negative charge (pKa ~2.6), it will likely interact with Q9NRA2-F1) (42). As expected, the N-domain and C-domain of the R168 side chain and thus break the E171-R168 interaction. the predicted structure could be superimposed with their counter- Then, the free E171 side chain can be easily protonated. As the parts in our structure well with an RMSD of 0.737 and 0.698 Å, re- sialic acid moves down and reaches the substrate-binding pocket, spectively (fig. S13A). However, as a whole, the two structures do Sialin changes from state 2 to state 3. Sialic acid interacts with + not overlap with each other, indicating that AF-Q9NRA2-F1 may R57 and breaks the E175-R57 interaction to free E175. Here, H adopt a different conformation. We analyzed the central pore in on the E171 side chain can be freely transferred onto E175 AF-Q9NRA2-F1 by MoleOnline and attempted computational because of the proximity of the two residues (one helical turn docking with Autodock Vina. The result shows that (i) the away). The deprotonated E171 restores its interaction with R168, Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 5of9

SCIENCEADVANCES | RESEARCHARTICLE MATERIALSANDMETHODS Protein expression and puri昀椀cation Thefull-lengthhumanSialin(UniProt:Q9NRA2)wasclonedintoa pFastBacvector(ThermoFisherScientific)withanN-terminalHis- tag and a thrombin digestion site. Three predicted N-linked glyco- sylation sites were abolished by site-directed mutagenesis (N71A, N77A, and N95A). Sialin was overexpressed in High Five cells using the Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific). Cells were harvested by centrifugation 72 hours after virus infection and lysed by passing through a microfluidizer M110P (Microfluidics Corporation). The membrane fraction was collected by centrifugation at 150,000g for 1 hour and resuspended inbufferA[20mMtris(pH7.4)and150mMNaCl].Laurylmaltose neopentylglycol(LMNG;1%)(Anatrace)wasusedtosolubilizethe membraneat4°Cfor2hours.Thesupernatantwasisolatedbycen- trifugation at 150,000g for 1 hour and incubated with TALON IMAC resin (Clontech) in buffer A with 5 mM imidazole. The resin was washed with buffer B [20 mM tris (pH 7.4), 150 mM NaCl, and 0.003% LMNG] with 10 mM imidazole, and then the protein was eluted in buffer B with 200 mM imidazole. The Fig. 5. Proposed model of sialic acid/H+ cotransport by Sialin. In the outward- eluted protein was digested with thrombin (Enzyme Research Lab- facing conformation (state 1), R168 interacts with E171, and R57 interacts with oratories)atamolarratioof1:50overnightat4°C.Sialinwasfurther E175. When the sialic acid is recognized (state 2), it likely interacts with R168 purified by gel filtration chromatography with a Superdex 200 and frees E171 to be pronated. As the substrate moves down the translocation column (Sigma-Aldrich) in buffer B and concentrated to ~5 mg/ pathway (state 3), it interacts with R57 and frees E175. E171 then transfers the ml for storage. H+ to E175. As E171 rebonds with R168, the substrate moves further down. In the inward-facing conformation (state 4), since E175 prefers a deprotonation Fab generation state (pK ~3.0), the H+ is quickly released to the cytosol. E175 rebonds with a Monoclonal antibodies against Sialin were generated in mice using R57, which promotes the substrate release from the binding pocket. Sialin is purified protein in detergent as the antigen in the Monoclonal An- then reset to the outward-facing conformation for another cycle. tibody Core Laboratory at the Oregon Health & Science University destabilizing the transient R168–sialic acid interaction and promot- (45). Antibody 8B1 was selected as it had the strongest binding af- ing the R57–sialic acid interaction in the pocket. Next, Sialin will finity to Sialin when tested by Western blot andELISA.TheFabwas change from state 3 to state 4 to release both H+ and sialic acid produced by papain digestion and purified by protein A affinity + chromatography (Thermo Fisher Scientific). into the cytosol. For the transferred H , the release process shall be easy because E175’s low pK favors a deprotonated state under a Cryo-EM sample preparation cytosolic pH. For sialic acid, the release process may be complicated ToovercomethechallengepresentedbythesmallsizeofSialin(~55 andinvolvemanyresiduesaroundthebindingpocket.Forexample, kDa), we first reconstituted the purified protein into lipid nano- polar residues, such as N430, N302, Y54, and Y119 (Fig. 2D), may discs. We then formed a larger complex with Fab generated from provide electrostatic force to move the sialic acid away from R57. the 8B1 antibody. Briefly, membrane scaffold protein MSP1D1 Thesugarringinsialicacidcouldinteractwithnearbyhydrophobic (Addgene) was purified as described previously (46). Purified residues. In addition, deprotonated E175 will likely rebind R57. All Sialin in detergent was mixed with MSP1D1 and soybean polar these possible interactions probably act coordinately to remove the extract lipids (Anatrace) at a molar ratio of 1:8:400 on ice for 30 sialic acid from the substrate-binding pocket. Last, Sialin could reset min. Prewetted Bio-Beads (0.2 mg; Bio-Rad Laboratories) was itself to state 1, the outward-facing conformation. The structure de- added for overnight incubation at 4°C. The mixture was purified termined here represents Sialin at state 3 but without sialic acid. by gel filtration with a Superose 6 column in buffer A, incubated In summary, our structure provides an excellent start to under- with Fab at a molar ratio of 1:2, and then purified by gel filtration. + standing the sialic acid/H cotransport mediated by Sialin. Thefinal complex of Sialin-nanodisc-Fab was concentrated to ~2.5 However, there are still many open questions regarding the molec- mg/ml. ular mechanism. Forexample, how does Sialin open the H183 con- striction site to release the substrate? How does the transporter Cryo-EM data collection switch from inward-facing to outward-facing? Future studies in bi- Three microliters of the freshly purified complex was applied to a ophysics, structural biology, and molecular dynamics simulations plasma-cleanedC-flatholycarbongrid(1.2/1.3,400mesh,Electron will be necessary to answer these questions. Microscopy Sciences) and prepared using a Vitrobot Mark IV (Thermo Fisher Scientific) with the environmental chamber set at 100% humidity and 4°C. The grid was blotted for ~3 s and then flash-frozen in liquid ethane. The data were collected on a Titan Krios (ThermoFisherScientific) operated at 300 keVand equipped Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 6of9

SCIENCEADVANCES | RESEARCHARTICLE with a K3 direct detector (Gatan). A total of 10,235 movies were re- membranefilterusingaminiextruder(AvantiPolarLipids).Tore- corded with a calibrated pixel size of 0.826 Å, a defocus range of constitute proteoliposomes, the liposomes were destabilized by 2 −1to−2.5μm,and50frameswithatotaldoseof~60electrons/Å . 0.015% Triton X-100 and then incubated with purified Sialin at a ratio of 150:1 (wt/wt) for 20 min at room temperature. Bio-Beads Image processing, model building, and re昀椀nement SM-2 (150 mg; Bio-Rad Laboratories) were added to the mixture The data were processed with cryoSPARC (47). Patch motion cor- to absorb all detergents overnight at 4°C. The reconstituted proteo- rectionwasusedtocorrectthebeam-inducedmovement,andpatch liposomes were harvested by ultracentrifugation at 180,000g for 20 contrast transfer function was used to estimate contrast transfer minandthenresuspended in buffer D [20 mM MES (pH 5.6) and function parameters for each movie. A total of ~3 million particles 150mMNaCl].Proteoliposomesolution(100μl)foreachtransport 3 were automatically picked using “Blob Picker” and extracted with a was incubated with 10 nM [ H]Neu5Ac at room temperature. At box size of 320 × 320 pixels. After two rounds of reference-free 2D different time points, the proteoliposomes were filtered and classification, ab initio reconstruction, and heterogeneous refine- washed with ice-cold Buffer A. The filter membrane was scintilla- ment, ~800,000 particles were selected for further processing. 3D tion-counted. Each transport was repeated five times. The trans- classification without alignment was performed with the following ported amount was calculated with the assumption that the parameters: five classes, 6-Å target resolution, and principal com- orientation of reconstituted Sialin was 50:50. ponent analysis initiation mode. The final reconstruction was ob- tained with a particular class of 394,078 particles. After Immuno昀氀uorescence staining and microscopy imaging homogenesis refinement and nonuniform refinement, the map High Five cells with or without His-tagged Sialin expression were reached a resolution of ~3.7 Å. A soft mask around Sialin and fixed in 2% paraformaldehyde for 30 min at room temperature. half of the Fab was generated for a final round of local refinement After washing three times with PBS, cells were permeabilized with to produce the 3.4-Å-resolution map. The model was built in Coot 0.2% Triton X-100 for 30 min. Unpermeabilized or permeabilized (48). The AlphaFold-predicted model of Sialin was used as a guide cells were blocked in PBS containing 10% horseserumfor30minat together with several secondary structure prediction programs: roomtemperature.Cellswerethenincubatedwithprimaryantibod- Jpred (49) and PSSpred (50). The final model contains residues ies against 6×His (1:500; UBPBio) or anti-Sialin monoclonal anti- 32 to 68 and 102 to 488, missing flexible N-termini, C-termini, body 8B1 (1:2000) in 5% horse serum in PBS for 1 hour at room and the luminal loop between TM2 and TM3. The model was temperature. Cells were washed three times in PBS before costained refined in PHENIX (51). The quality of the model was assessed withsecondaryantibody(1:800;MolecularProbesAlexaFluor555) by MolProbity (52). Statistical details can be found in table S1. All andnucleidyeHoechst(1:5000;MolecularProbes)or4′,6-diamidi- superpositions of structures were calculated in the program UCSF no-2-phenylindole (Sigma-Aldrich; 1 μg/ml) for 1 hour at room ChimerawiththealignmentalgorithmofNeedleman-Wunschand temperature.StainedcellswerewashedthreetimesinPBS,followed BLOSUM-62matrix (53). by microscopy imaging with an Olympus FV1000 FCS/RICS con- focal microscope or a Keyence BZ-X710 All-in-One Fluorescence Cellular transport assay Microscope. Sialic acid uptake was measured in High-Five cells expressing wild- type and various mutations of Sialin. Specifically, 200 μl of High- Five cells (0.4 × 106 count/ml) was seeded in 24-well plates in Supplementary Materials Grace’s Insect Medium supplemented with fetal bovine serum This PDF 昀椀le includes: and infected with the corresponding baculoviruses for 24 hours. Figs. S1 to S13 Cells were washed twice with buffer C [20 mM MES (pH 5.6), 5 Tables S1 and S2 mMglucose,150mMNaCl,and1mMMgSO ]andthenincubated References 3 4 with[ H]Neu5Ac(0.05μCi,assumedtobe10nMin250μlofbuffer View/request a protocol for this paper from Bio-protocol. C) for 15 min at room temperature. After washing twice with ice- cold buffer at pH 7.4, the radioactivity in the cells was counted by liquid scintillation counting using a PerkinElmer Tri-Carb 2910 TR REFERENCESANDNOTES machine. Each transport was measured five times independently. 1. D. Adams, M. Wasserstein, in GeneReviews((R)), M. P. Adam, H. H. Ardinger, R. A. Pagson, The expression level of wild-type and mutant Sialin in High-Five S. E. Wallace, L. J. H. Bean, K. W. Gripp, G. M. Mirzaa, A. Amemiya, Eds. (University of cells was determined by Western blot analysis, which was used to Washington, Seattle, 1993). normalize the substrate transport measurements. The transported 2. T. Angata, A. 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N.J.Myall,C.C.Wreden,M.Wlizla,R.J.Reimer, G328EandG409Esialinmissensemutations chepain,B.Gasnier,C.Anne, Aminoacidsbearingaromaticorheteroaromaticsubstituents similarly impair transportactivity,butdi昀昀erentiallya昀昀ecttra昀케cking.Mol.Genet.Metab. 92, as a new class of ligands for the lysosomal sialic acid transporter sialin. J. Med. Chem. 63, 371–374 (2007). 8231–8249 (2020). 32. O. Trott, A. J. Olson, AutoDock Vina: Improving the speed and accuracy of docking with a Acknowledgments: We thank the sta昀昀 in the Paci昀椀c Northwest Center, especially newscoring function, e昀케cient optimization, and multithreading. J. Comput. Chem. 31, T. Humphreys,for the cryo-EM datacollection. A portion of this research was supported by NIH 455–461 (2010). grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 8of9

SCIENCEADVANCES | RESEARCHARTICLE (grid.436923.9), a DOE O昀케ce of Science User Facility sponsored by the O昀케ce of Biological and depositedintheProteinDataBank(https://www.rcsb.org/)undertheaccessioncode8DWI.All Environmental Research. We also thank J. Kieft for the critical reading and suggestions. data needed to evaluate the conclusions in the paper are present in the paper and/or the Funding:ThisworkwassupportedbyNIH(R01GM126626,R21AG064572,R01HL133230,and Supplementary Materials. R01HL159086). Author contributions: W.H., C.C., K.S., and H.Z. designed the experiments, collectedandanalyzedthedata,andwrotethemanuscript.Competinginterests:Theauthors Submitted 11 September 2022 declare that they have no competing interests. Data and materials availability: The cryo-EM Accepted 16 December 2022 mapofSialin was deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/ Published 20 January 2023 emdb/) under the accession code EMD-27755. Coordinates of the atomic model were 10.1126/sciadv.ade8346 Huetal., Sci. Adv. 9, eade8346 (2023) 20 January 2023 9of9