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