Samstag, Juli 30, 2022
StartBiochemistryCryo-EM construction of human glucose transporter GLUT4

Cryo-EM construction of human glucose transporter GLUT4

Exercise characterization and cryo-EM evaluation of GLUT4

The complete-length wildtype human GLUT4 was fused with an N-terminal Flag tag and transiently expressed in HEK293F cells. After sequential purification by way of anti-FLAG affinity resin and size-exclusion chromatography (SEC) (Supplementary Fig. 2a), the transport exercise of the height fraction was examined in a proteoliposome-based counterflow assay13,27. GLUT4 has a nominal Okaym of 5.4 mM and Vmax of three.7 µmol/mg/min for D-glucose transport (Supplementary Fig. 2b). CCB inhibits the glucose transport exercise of GLUT4 with an IC50 of three.7 µM (Supplementary Fig. 2c).

As analyzed above, GLUT4, which has a two-fold pseudo symmetry within the membrane and lacks a cumbersome soluble area, introduced many challenges for cryo-EM evaluation. Our earlier work with MCT126 and the Scap/Insig-2 advanced22,23 means that the scale of the micelles might have a direct impression on 2D and 3D classifications. We due to this fact got down to display screen for detergents that gave rise to the perfect protein sign (Fig. 1).

We utilized GLUT4 to a Superdex 200 column that was pre-equilibrated within the presence of the next detergents, 0.02% (w/v) glyco-diosgenin (GDN), 0.02% (w/v) n-dodecyl-β-D-maltoside (DDM) plus 0.002% (w/v) cholesteryl hemisuccinate tris salt (CHS), 0.01% (w/v) lauryl maltose neopentyl glycol (LMNG) plus 0.001% (w/v) CHS, and 0.1% (w/v) n-nonyl-β-D-glucopyranoside (β-NG). Certainly, totally different detergents gave rise to distinct elution peak volumes, earliest at 15 ml with GDN and newest at 15.7 ml with β-NG (Fig. 1c). We additionally efficiently reconstituted the purified GLUT4 into nanodiscs of POPC plus ldl cholesterol surrounded by the membrane scaffold protein MSP1D1. The elution quantity of nanodisc-embedded GLUT4 is barely sooner than β-NG (Fig. 1c).

Subsequent, we examined the proteins purified in numerous situations utilizing cryo-EM. To stabilize the construction of GLUT4, 1 mM CCB was added to the purified proteins. Particulars for cryo-sample preparation and cryo-EM information acquisition are introduced intimately in Strategies. From the 2D class averages, it’s instantly clear that GDN or DDM/CHS leads to a weaker protein sign with comparatively massive and thick micelle. The opposite three situations, β-NG, LMNG/CHS, and nanodisc, all current smaller measurement, and options attribute of the transmembrane segments (TMs) are discernible (Fig. 1d). We, due to this fact, proceeded with these three situations for 3D reconstruction.

For structural willpower of small membrane proteins, an excellent reference map is essential for additional classification and refinement. Utilizing Ab-initio development in cryoSPARC28, we have been capable of acquire a good preliminary mannequin for the dataset of GLUT4 in β-NG (Supplementary Fig. 3), however not for that in LMNG/CHS or nanodisc. Particulars might be present in Strategies. Following our beforehand developed “guided multi-reference 3D classification” and “seed”-facilitated 3D classification technique22,23,26,29, the resolutions of GLUT4 embedded within the LMNG/CHS micelles and nanodiscs each reached 3.3 Å after a number of rounds of 3D classification and refinement by cryoSPARC (Fig. 1e, Supplementary Figs. 4, 5 and Supplementary Desk 2). The superb EM map allowed for project of 464 aspect chains within the 12 TMs and the intracellular helical (ICH) area (Fig. 1f, Supplementary Fig. 6, and Supplementary Desk 2).

Inward-open construction of GLUT4 sure to CCB

Within the construction of GLUT4, the amino terminal area (NTD, containing TMs 1-6) and carboxyl terminal area (CTD, containing TMs 7-12) enclose a big cavity that opens to the intracellular aspect, a state outlined as inward-open (Fig. 2a, left). The inhibitor CCB is thought to inhibit GLUTs with inward-open conformation30,31, which is in step with our current construction. CCB is accommodated within the large cleft between the NTD and CTD (Fig. 2a, proper higher panel). CCB includes three ring constructions, a macrolide ring, a nine-membered bicyclic ring, and a phenyl ring, all of that are well-resolved within the cryo-EM map (Fig. 2a, proper decrease panel).

Fig. 2: Inward-open construction of GLUT4 sure to the endofacial inhibitor CCB.
figure 2

a Cryo-EM construction of GLUT4 in an inward-open state. Two perpendicular views of area coloured GLUT4 are proven. CCB, proven as inexperienced ball-and-sticks, binds to the central substrate binding website that’s open to the intracellular aspect. The glycan on the extracellular aspect is proven as inexperienced sticks. The EM density for the sure CCB, proven as inexperienced mesh, is contoured at 4 σ. b Polar interactions between CCB and GLUT4. Key residues for inhibitor coordination are proven as sticks. The cyan dashed strains point out hydrogen bonds. c Hydrophobic residues surrounding CCB. Two perpendicular views are proven. d A schematic presentation of CCB coordination by GLU4. Van der Waals contacts are indicated by black dashed curves, and hydrophilic interactions are indicated by cyan dashes.

The macrolide ring of CCB is coordinated by Asn176 and Trp404 by way of polar interactions, and Phe38, Trp404, and Trp428 by way of hydrophobic interplay (Fig. 2b, c). The bicyclic ring is nestled in a hydrophobic cavity constituted by Ile42, Ile180, Ile184, Ile303, Phe307, and Phe395. The coordination is buttressed by hydrogen bonds with Gln298, Gln299, and Trp404. The phenyl ring interacts with Ile180, Gln177, and Pro401 by way of hydrophobic contacts (Fig. 2b–d).

Comparability of inward-open GLUT4 and GLUT1

We subsequent in contrast GLUT4 to GLUT1, whose constructions have additionally been captured within the inward-open state within the presence of CCB (PDB code: 5EQI) or β-NG (PDB code: 4PYP)12,31. The cryo-EM construction of GLUT4 might be superimposed to the crystal constructions of GLUT1-CCB and GLUT1-NG with RMSD values of 1.13 Å and 1.09 Å over 439 and 435 aligned Cα atoms, respectively, with similar transmembrane domains (Fig. 3a). Amongst all of the CCB-coordinating residues, two loci are totally different between the 2 proteins; Ile42 and Asn176 in GLUT4 are respectively substituted with Thr30 and His160 in GLUT1. The phenyl ring of CCB rotates by about 60 levels within the two constructions (Fig. 3b).

Fig. 3: Structural comparability of inward-open GLUT4 and GLUT1.
figure 3

a Structural comparability of GLUT4 and GLUT1. Proven listed here are superimposed constructions of GLUT4 (area coloured) with GLUT1 sure to CCB (silver, PDB code: 5EQI) or within the presence of β-NG (pink, PDB code: 4PYP). The TM area stays practically similar. b Related coordination of CCB by GLUT4 and GLUT1. Amongst all of the CCB-coordinating residues, there are solely two diversified residues, Ile42 and Asn176 in GLUT4 are respectively substituted with Thr30 and His160 in GLUT1. c Cryo-EM evaluation reveals the glycosylation website on GLUT4. The density for 2 sugar moieties (inexperienced) connect to Asn57 on the extracellular helix TM1e of GLUT4 is contoured at 4 σ. d Deviations of the ICH area between GLUT4 and GLUT1. In distinction to the practically similar constructions of the TM area, there are deviations of ICH3 and ICH4 between GLUT4 and GLUT1. Extra importantly, the C-terminal ICH5 is resolved in GLUT4 solely.

GLUT1-4 all include a conserved N-linked glycosylation website on an extracellular helix designated TM1e, which is a bent extension of TM1. To facilitate crystallization, the glycosylation website in GLUT1, GLUT3, and GLUT5 was eradicated by single level mutation or deglycosylation therapy12,13,31,32. Within the cryo-EM map for GLUT4, a attribute glycan density is noticed contiguous with Asn57, exemplifying the distinctive energy of cryo-EM in resolving posttranslational modifications (Fig. 3c).

Apart from the remark of the extracellular glycosylation, ICH displays essentially the most evident variations between GLUT4 and GLUT1. Our earlier structural evaluation of GLUT1 and GLUT3 outlined 5 ICH helices, ICH1-ICH4 between the N and C domains and ICH5 after TM1212,13. ICH3 and ICH4 show minor positional shifts between GLUT4 and GLUT1 (Fig. 3d). The most important distinction happens to ICH5, which is invisible within the construction of GLUT1, however clearly resolved within the EM map of GLUT4 (Fig. 3d and Supplementary Fig. 6). We examined the practical position of ICH5 primarily based on the structural comparability of outward-facing GLUT3 and inward-open GLUT4.

ICH5 is essential for the transport exercise of GLUTs

The ICH area undergoes main rearrangement between the outward-facing construction of GLUT3 and inward-open construction of GLUT4 (Fig. 4a). Within the construction of GLUT3 (PDB code: 4ZW9), the ICH area serves as a latch to safe the closure of the N and C domains on the intracellular aspect. Phe458 on ICH5 is surrounded by three Arg residues, Arg151 on TM5, Arg210 on ICH1, and Arg398 on TM11 (Fig. 4b, left). These residues are invariant between GLUT3 and GLUT4 (Supplementary Fig. 1). As ICH5 was invisible in earlier constructions, we didn’t get an opportunity to look at the rearrangement of this cation-π cluster. Within the cryo-EM map for GLUT4, Phe476 (comparable to Phe458 in GLUT3) is clearly resolved (Supplementary Fig. 6). The cation-π community is totally disassembled with ICH5 shifting away (Fig. 4b, proper).

Fig. 4: The ICH area contributes to the intracellular gating throughout alternating entry cycle of GLUTs.
figure 4

a Conformational adjustments of the ICH area between the outward-occluded GLUT3 and inward-open GLUT4 constructions. The cryo-EM construction of GLUT4 is superimposed to the crystal construction of GLUT3 (PDB code: 4ZW9) relative to the N (left) and C (proper) domains, respectively. b A conserved Phe residue on ICH5 serves as an area organizing middle within the outward-facing state. left, Phe458 on helix ICH5 types cation-π interactions with a number of Arg residues within the N and C domains of outward-facing GLUT3. proper, Rearrangement of the ICH area in inward-open GLUT4. Phe476 in GLUT4 (equal of Phe458 in GLUT3) now not interacts with any of the Arg residues within the inward-open state. c The interplay between Phe458 and N-domain Arg residues contributes to the transport exercise of GLUTs. Proven listed here are normalized transport actions of GLUT4 variants within the liposome-based counterflow assay. Information are introduced as imply with commonplace deviation, in three impartial experiments. Supply information are supplied as a Supply Information file.

To look at the practical relevance of this intracellular cluster, we substituted every of the Arg residues and Phe476 of GLUT4 with Ala and carried out counterflow assay for these variants (Fig. 4c). GLUT4-F476A solely retained half of the transport exercise of WT, supporting an essential position of this ICH5 residue within the alternating entry transport strategy of GLUT4. Ala alternative of Arg169 (equal of GLUT3-Arg151), which additionally interacts with a Glu residue on TM10 and two spine carbonyl oxygen teams on the loop between TM12 and ICH5, misplaced greater than 70% of the exercise. Mutation of Arg228 (equal of GLUT3-Arg210), which is principally concerned in bridging ICH1 and ICH3 aside from binding to Phe476, lowered the exercise by ~40%. Mutation of Arg416 has the least impact on the transport exercise, with a discount of roughly 20%. This result’s in step with the C domain-localization of Arg416, as there’s barely any relative movement between the C area and ICH5 through the alternating entry cycle (Fig. 4a). The assay outcomes are in step with the structural indication that ICH5 is essential for the transport exercise of GLUTs.


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