Research Progress on the Structure and Function of Glucose Transporter GLUT Family
1. Overview and Biological Significance of GLUT Family
The glucose transporter (GLUT) family, as an important member of the major facilitator superfamily (MFS), is currently known to be the largest class of membrane transport protein families. These proteins play a crucial physiological role in transporting glucose into cells. Given that glucose is the fundamental energy source for most organisms, GLUT family proteins are irreplaceable in maintaining metabolic balance, making them a long-term focus in structural biology and metabolic research.
From a molecular evolution perspective, GLUT family proteins exhibit typical MFS superfamily structural characteristics. In 2014, Yan Ning's research team made significant breakthroughs by successfully resolving the high-resolution crystal structure of human glucose transporter GLUT1 for the first time. This pioneering study revealed that GLUT1 consists of two domains: an N-terminal domain and a C-terminal domain formed by 12 classic transmembrane helices; it also unexpectedly discovered a unique intracellular soluble region composed of four α-helices (ICH). Notably, this unique structural sequence has only been observed within the sugar transport subfamily of MFS members, suggesting its potential special function during carbohydrate transport.
2. Milestone Advances in Structural Research on GLUT Family
In 2015, another major breakthrough occurred in structural biology when Yan Ning's team successfully resolved high-resolution structures representing three different conformational states of GLUT3 using X-ray crystallography techniques. In stark contrast to previously obtained inward-open conformations for GLUT1, these newly resolved structures exhibited distinctly different outward-open and outward-closed conformations for GLUT3. This study provided comprehensive insights into how substrate transport occurs through conformational changes within glutamate family proteins at a molecular level.
In May 2022, new advancements were achieved in this field with innovative use of lipid cubic phase (LCP) crystallization technology by Yan Ning’s group to resolve complex crystal structures between compound SA47 and GLUT3. This result laid an essential structural foundation for developing novel inhibitors targeting surfaces on GLU T proteins. Meanwhile, Tsinghua University’s Yan Chuangye team collaborated with Yan Ning’s group to publish research regarding frozen electron microscopy structure studies on GLUT4 in Nature Communications journal—this study systematically compared various detergent conditions alongside nanodisc technology obtaining human-derived GLU T4 combined with small molecule inhibitor cytochalasin B (CCB), ultimately achieving resolution up to 3.3Å . These groundbreaking works greatly deepened our understanding concerning interaction mechanisms between GLU T family proteins along with inhibitors.
3.Structural Features & Functional Significance Of G LUT4
As one primary glucose transporter found predominantly within adipocytes as well as skeletal muscle cells ,GLUT4 plays critical roles during insulin-regulated uptake processes . From physiological perspectives ,obstruction occurring while transferring membranes viaGLUT4is regardedasoneofimportantmolecularbasesforinsulinresistanceorType2diabetesdevelopment.Underbasicphysiologicalconditions ,GLUT4mainlydistributesacrossthetrans-Golgi network(TGN),endosomes,and specializedGLUT4storagevesicles(GSVs).Uponinsulin stimulation ,thetransportationofGL UTquicklyshiftsfromtheseintracellularstructuresintotheplasmamembrane,resultinginthefastuptakeofglucosefrombloodstream.Hence elucidatingfine structureandworkingmechanismsofG LUTnotonlyfacilitatesunderstandingfundamentalprinciplesenergy metabolismbutalsoofferscrucialcluesfordevelopingnewdrugsfortreatingmetabolicdiseases.
Frommolecularcharacteristics perspective,G LUT4encodedbySLC2A4genebelongs tothe solute carrier(SLC)transportproteinfamily.Notably,the SLCtransportproteinfamilyrepresents thesecondlargestclassofmembranetransporterswithin humangenomefollowingG-proteincoupled receptors(GPCRs).Among14membersbelongingtotheS LC2Asub-family,G LUT4sharesclosest evolutionaryrelationshipwithG LUT1,bothshowingsimilarityidentityandsimilarityratesreaching65%and79%,respectively.Nevertheless,G LUThassomeunique sequence featuresincludingN-terminalFQQImotifandC-terminalLL&TELEY motifswhicharenotconservedamongotherS LC2Amemberspotentiallyrelatingitspeculiarregulatorymechanismsduringmembranetransportprocesses..
Four.TechnicalChallenges&BreakthroughsinStructureAnalysisOf G LUT nTraditionalstructuralbiologymethodsfacedmajortechnicalbottleneckswhenstudyingG LUTFour.DespiteinitialattemptsatusingX-raycrystallographytorevealstructure,significanteffortsfailedtodevelophigh-qualitydiffracting crystals.Thispromptedresearchteamstoexplorefrozen-electronmicroscopytechnologieshowever facedmultiplechallengesinvolvedinstructurerevelation.G LUTFourconsistsof509aminoacidresidueswithsmallermolecularweight;itsN-andC-terminusexhibitingdualpseudo-symmetryperpendicular tomembranes;lackingdistinctsoluble regions;directionalityindetergentormicellewaschallenging.AllthesefactorscombinedrenderedG LUTFouranextremelychallengingobjectoffrozen-electronmicroscopyanalysis.Toovercome thesetechnicalissues,researchteamsystematicallyoptimizedexperimentalprotocols.Firstcarefullyscreeneddetergentconditionscomparedfourdifferentdetergentsystems(0 .02%GD N、0 .02%DDM+0 .002%CH S、0 .01%LM NG+0 .001%C HS及0 .1%β-N G )ontheeffectsonpurificationresults.ExperimentdatarevealedthatvariousdetergentsindeedproducedsignificantlydifferentelutionpeakswhereinGD Nsolutionpeakappearedat15mlwhileβ-N G peakoccurredlastat15．7ml.Meanwhile，theteamalsosuccessfullyreconstitutedpurifiedG L UTFourintoananano-disk systemencapsulatedby membrane scaffold proteinID P which showedearlier elution peak than β-N G condition.Frozen electron microscopyobservationsdemonstratedthatmicellesformedvia GD Nor DDM + CH S yieldedlarger thicker sizes resultingweakersignalsoftheprotein whereasothershowcasedsmaller dimensionswithcleartransmembranefeatures.Finally,purifiedthroughβ-N Greachedresolutionupto41 Å whilstbothLMNG/CH Sandnanodiskconditionsachieved33 Åresolution.These technologicalbreakthroughsofferimportantmethodologicalreferencestructureanalysesforsmall-membraneproteins.. n n ### Five.DetailedStructuralFeatureAnalysisOfGlutFourHighResolutionStructuresdisplaytypicalinternalopenconformation.Nterminaldomain(includingTM helicesI-VIshowninblue)alongsideCTD(includingTM helicesVII-XIIshowninviolet)togetherformalargecavityfacinginsidecell.InhibitorCCBpreciselylocatesbetweenNTD&CTDlargegapcomposedthreecyclicstructures:alargering,a9-memberdouble-ringanda phenylring.StructureanalysisunveilsinteractionsdetailsbetweenCCBandG LTFOUR：large ring forms polar interactionswithN176&W404whilsthydrophobicinteracts occurviaF38,W404&W428；double-ring fits inside hydrophobic cavity constitutedby I180,I42,I184,F307,I303&F395heldstable hydrogenbondnetworkprovidedbyQ298,Q299&W404；phenylring interacts throughhydrophobiccontactsagainstI180,Q177&P401.Comparativeinvestigationbetweenthestructureoftwo reveals highlysimilarity across their transmembraneregionsalongsideidenticalcooperativeinteraction patterns againstCCB.Amongall residues interacting w ith CCB,two sites differ : I42(N176correspondstoThr30&H160 respectively); moreover,C CBphenylring exhibitsapproximately60-degree rotationaldifference betweenthestructures.Particularly noteworthy wasfirstobservationoftypically glycosydensityadjacenttoN57withinGlutFourhighlightinguniqueadvantagefrozelectronmicroscopytechniques decipherpost-translationalmodifications involvingproteins...\ ...
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