Main

IP6 is a highly negatively charged compound that is present in all mammalian cells at concentrations of 10–40 µM6. Inositol phosphates stimulate in vitro assembly of HIV-1 Gag into immature virus-like particles (VLPs), with previous data suggesting that IP6 interacts with both the MA and NC domains of Gag7,8,9. To understand how IP6 affects HIV-1 assembly, we used an HIV-1 Gag construct spanning the CA to NC domains and having one extra amino acid residue, Ser, preceding the normal N-terminal Pro at the start of the CA domain (s-CANC; Fig. 1a), because this should disfavour formation of the N-terminal β-hairpin that promotes mature assembly10. Longer N-terminal extensions of CANC constructs have been shown to assemble inefficiently into immature VLPs at pH 8, but into mature VLPs at pH 61,10. However, we found that s-CANC still formed mature-like particles at both pH values (Fig. 1b). Notably, the presence of IP6 induced a marked switch to the formation of spherical, immature VLPs (Fig. 1b, d). At pH 8, even a substoichiometric 1:50 molar ratio of IP6 to protein resulted in an approximately 100-fold increase in immature VLPs (Fig. 1b, c). At pH 6, the effect of IP6 was less strong, requiring at least a 1:10 ratio to induce immature assembly (Fig. 1b). We conclude that IP6 imposes an in vitro immature assembly phenotype, even under conditions that favour the mature lattice (pH 6).

Fig. 1: IP6 induces assembly of HIV-1 Gag in vitro.
figure 1

a, Map of the HIV-1 Gag protein, indicating the MA, CA, NC and p6 domains, and spacer peptides SP1 and SP2. Gag-derived constructs used in this study are shown underneath. Blue bar, major homology region; purple bar, SP1 helix; NTD and CTD, N-terminal and C-terminal domains of CA; R18, K290 and K359, locations of mutations; N372, C-terminal residue of the s-CASP1 and CACTDSP1 constructs. b, Negative-stain electron microscopy images of mature and immature VLPs formed by s-CANC (50 μM) at pH 6 and pH 8 in the absence or presence of the indicated molar ratios of IP6 (0–10 μM). Scale bars, 100 nm. c, Number of VLPs per 55 µm2 without (–) and with (+) 10 µM IP6 at pH 6 and pH 8; n shown below and mean above box plots. The experiment was repeated three times with similar results. d, Diameters of immature and mature VLPs; n shown below and mean above box plots. e, Representative images of s-CANC VLPs assembled at pH 8 in the absence and presence of IP3, IP4, IP5 and IP6. Scale bars, 100 nm. The experiment was repeated twice with similar results. f, Number of VLPs per 55 µm2 without and with 10 µM IP3–IP6; n = 5, mean shown above box plots. g, Parallel transfections of 293FT wild-type (WT) and IPPK knockout (KO) cells were performed with a VSV-G-pseudotyped HIV-1 provirus containing GFP, and infectivity was measured on WT 293T cells. Graphs show mean ± s.d. of four independent experiments; dots show individual data points. Right panels show sequences of total PCR products of the guide RNA target sites from WT and KO cells; guide RNA sequence is underlined in red. c, d, f, Centre lines show medians; box limits indicate 25th and 75th percentiles as determined by R software24; whiskers extend to minimum and maximum values.

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Other inositol derivatives also promoted s-CANC assembly, but to a lesser extent, in the order IP3 < IP4 < IP5 < IP6 (Fig. 1e, f), with efficacy correlating with the number of phosphate groups. Other negatively charged compounds did not promote or only marginally promoted assembly (Extended Data Fig. 1). Overall, these results indicate that charge neutralization is a fundamental aspect of IP6-mediated HIV-1 Gag assembly, and that the details of coordination geometry and/or local stereochemistry are also important.

To address the biological importance of IP6 in HIV-1 replication, we generated a knockout cell line in which the gene encoding inositol pentakisphosphate 2-kinase (IPPK), the enzyme responsible for the final step in IP6 synthesis, was ablated (Fig. 1g). Infectious HIV-1 particle production from these knockout cells was reduced by between 10- and 20-fold (Fig. 1g). We interpret this result as implying that IP6 has a critical role in assembly of immature and/or mature HIV-1.

As the s-CANC construct lacks the MA domain, the effect of IP6 cannot depend on this domain, as previously suggested7,8,9. The NC domain also cannot be essential, because IP6 still promoted assembly in the absence of nucleic acid (Extended Data Fig. 2a, d). Furthermore, IP6 also promoted the formation of abundant immature VLPs from the smaller protein s-CASP1, which lacks the NC domain altogether (Fig. 2a and Extended Data Fig. 2b, d). However, deletion of the SP1 domain abrogated the effect of IP6, as IP6 failed to induce assembly of s-CA into immature VLPs (Extended Data Fig. 2c).

Fig. 2: IP6 interacts with Lys290 and Lys359 in the immature HIV-1 Gag hexamer.
figure 2

a, IP6-induced assembly of s-CASP1 into immature VLPs. The experiment was repeated four times with similar results. b, IP6-induced assembly of CACTDSP1 into flat micro-crystals. The experiment was repeated six times with similar results. c, Two-dimensional cryo-EM projection map of a micro-crystal. Images of multiple crystals were collected during two rounds of data collection from separate assembly reactions and all crystals had similar unit cells. Two individual crystals had single layer regions and could be further processed. These crystals generated similar maps. d, e, Top view (d) and side view (e) of the CACTDSP1 hexamer X-ray crystal structure showing the protein in grey ribbons and unbiased mFo–DFc difference density in blue mesh, contoured at 2σ. f, Top and side views of IP6 in its myo configuration, docked into the difference density as a rigid body in one of six rotationally equivalent orientations. All six binding modes are shown in Extended Data Fig. 4a. g, Side view of the two rings of Lys290 (green) and Lys359 (cyan) with bound IP6 in the middle. Densities were omitted for clarity, and are shown in Extended Data Fig. 4b.

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Both the CA domain of Gag and the mature CA protein are composed of two separately folded sub-domains, CANTD and CACTD. To further define the site of action of IP6, we removed the N-terminal CANTD sub-domain to create CACTDSP1, which makes up the minimal Gag hexagonal lattice3,11. In the presence but not the absence of IP6, and at physiological pH and ionic strength, CACTDSP1 formed flat hexagonal crystals, as shown by negative-stain electron microscopy (Fig. 2b). These crystals had the characteristic immature lattice spacing (Fig. 2c). That s-CASP1 formed a spherical lattice while CACTDSP1 formed a flat lattice suggests that CANTD provides the contacts necessary for enforcing lattice curvature.

We next determined the X-ray crystal structure of CACTDSP1 crystallized in the presence of IP6 (Fig. 2d, e and Extended Data Table 1). This revealed a single, six-fold symmetric density in the middle of the hexameric ring (blue mesh in Fig. 2d–f), indicating that one IP6 molecule binds one CACTDSP1 hexamer. Notably, this density coincides precisely with an unknown density feature observed in cryo-electron microscopy (cryo-EM) maps of the HIV-1 Gag hexamer derived from authentic immature virions2 (Extended Data Fig. 3). This further supports the idea that IP6 is a cofactor of Gag assembly in cells and is a structural component of the HIV-1 particle.

IP6 is an asymmetric molecule with multiple stereoisomers, the most abundant of which is the myo form, with a chair configuration of one axial and five equatorial phosphate groups12; this is the most commonly observed stereoisomer in structures of various IP6-binding proteins13,14,15. In our CACTDSP1 structure, the IP6 density is also consistent with the myo form, with the axial phosphate pointing towards the six-helix bundle (6HB) (Fig. 2f). The bound ligand can adopt six energetically equivalent orientations, and the six-fold symmetric density is therefore the sum of these equivalent positions (Extended Data Fig. 4a). More importantly, the bound IP6 is surrounded by two rings of lysine sidechains—Lys290 from the major homology region loop and Lys359 from the 6HB (Fig. 2g). In our previous crystal structure of the CACTDSP1 hexamer in the absence of IP6, sidechain densities for these lysines were not visible, implying that these residues were highly flexible3. In the current structure, these sidechains are better ordered, and in direct ionic contact through their primary ε-amines with the IP6 phosphate groups (Fig. 2g and Extended Data Fig. 4b).

Consistent with the structure, we found that s-CANC mutant proteins in which Lys290 or Lys359 were replaced with alanine (K290A and K359A) were 100-fold less responsive to added IP6 (Extended Data Fig. 5a–d). These results further indicate that both lysine rings are required for productive IP6 binding. K290R and K359R mutants had less pronounced defects but still did not respond to IP6 as well as wild-type s-CANC, consistent with the high degree of lysine conservation in these positions (99.94% for K290 and 99.84% for K359; http://www.hiv.lanl.gov). Furthermore, the K290A and K359A mutations abolished infectivity (Extended Data Fig. 5e). Thus, optimal HIV-1 assembly in cells appears to require lysines at both positions. The results of previous studies that examined the effects of the above mutations on virus budding from cells and on virus infectivity are consistent with our findings16,17,18,19.

The above data suggest that IP6 acts by stabilizing the 6HB and promoting the formation of the immature Gag hexamer. To test this notion, we examined the dynamic behaviour of the CACTDSP1 hexamer by using all-atom molecular dynamics simulations. In the absence of IP6, the six-fold symmetry of the CACTDSP1 hexamer collapsed after 200 ns and did not recover during the 2 μs of simulation (Extended Data Fig. 6 and Supplementary Video 1). By contrast, six-fold symmetry in the presence of IP6 was maintained, particularly at the top of the 6HB, proximal to the IP6-binding site. Other inositol derivatives and mellitic acid (hexacarboxybenzene) also stabilized the 6HB in our simulations, consistent with their ability to also support immature s-CANC assembly in vitro (Extended Data Fig. 6b, c).

We also examined the effect of IP6 on mature capsid assembly, which is mediated by the CA protein that is generated upon Gag proteolysis. We found that IP6 promoted assembly of HIV-1 CA into mature-like structures1,10,20 (Fig. 3a and Extended Data Fig. 7b, d). Compared to immature s-CANC assembly, however, higher amounts of IP6 were required (Extended Data Fig. 7b, d). Mellitic acid (Extended Data Fig. 7c, e) and IP5, but not IP4 or IP3 (Extended Data Fig. 7f, g), stimulated mature CA assembly, although less potently than IP6.

Fig. 3: IP6 induces mature CA assembly by interacting with Arg18.
figure 3

a, Representative negative stain images of mature CA assemblies at pH 6 and 100 mM NaCl with increasing IP6 concentrations (0–1,250 μM). The experiment was repeated five times with similar results. b, Representative image of failed CA R18A assembly even in the presence of 1,250 μM IP6. The experiment was repeated three times with similar results. c, d, Top view (c) and side view (d) of a CA hexamer crystal structure showing the protein in yellow ribbons and unbiased mFo–DFc difference density in blue mesh, contoured at 2.2σ. e, Side views of myo-IP6 docked into the difference density in two possible binding modes. f, Illustration of a single IP6 molecule bound within a chamber enclosed by the N-terminal β-hairpins and the Arg18 ring (magenta). In a second crystal form, IP6 densities were observed both above and below the Arg18 ring (Extended Data Fig. 8).

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The mature HIV-1 CA hexamer also contains a positively charged ring, made up of Arg18 sidechains (Arg150 in Gag numbering)21,22. This ring was previously shown to mediate transport of nucleoside triphosphates, which facilitates reverse transcription of the encapsulated genome23. We therefore tested whether IP6 would promote assembly of the HIV-1 CA R18A mutant, and found that it did not (Fig. 3b). HIV-1 virions containing this mutation were also non-infectious17,23 (Extended Data Fig. 5e). We next crystallized the mature CA hexamer in the presence of IP6 (Extended Data Table 1). Although IP6 can bind both above and below the ring (Extended Data Fig. 8), densities were most pronounced in the upper binding site, inside a chamber surrounded by the N-terminal β-hairpins of CA (Fig. 3c–f). Thus, IP6 also binds and promotes assembly of the mature HIV-1 CA lattice.

Our results lead to the following model (Fig. 4). IP6 facilitates the formation of the six-helix CA–SP1 bundle by binding to two rings of primary amines at Lys290 and Lys359, thereby neutralizing otherwise repulsive charges at the centre of the HIV-1 Gag hexamer (Fig. 4c). Although other negatively charged molecules can also bind this pocket, our data suggest that IP6 is the most potent in promoting assembly, probably because it has the most optimal binding geometry. Some 300–400 molecules of IP6—one per hexamer—are incorporated into the virus particle as a structural component of the immature Gag shell (Fig. 4c). During virus maturation, proteolysis of Gag disrupts the 6HB, thus releasing IP6 and at the same time unmasking the Arg18 binding site in mature CA. IP6 then binds to this newly exposed site in CA (Fig. 4d), promoting the formation of CA hexamers and in turn the mature CA lattice. This involvement of a small molecule in two distinct steps in virus assembly, by binding to highly conserved sites, suggests strategies for possible therapeutic intervention in HIV-1 replication.

Fig. 4: Model.
figure 4

a, Diagram of HIV-1 Gag, with the indicated positions of R150 (triangle; R18 in mature CA), K290 (circle; K158 in mature CA), K359 (square; K227 in mature CA). Dotted lines indicate protease cleavage sites. b, Diagram of Gag organization in immature virions (left). Following cleavage of Gag by protease (that is, maturation), CA re-organizes to form a mature core around viral RNA (right). c, d, Surface representations of the CASP1 and CA hexamers in the immature (c) and mature virus (d), with IP6 shown in its binding sites. The marked rearrangement of CA upon maturation is evident, as is the change in IP6 binding site between immature and mature viruses. CANTD, blue; CACTD, orange; 6HB, purple; IP6, red.

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Methods

No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.

Protein purification

DNA coding for HIV-1 Gag proteins were cloned into a His6–SUMO vector25. The proteins were expressed in Escherichia coli and purified using standard Ni2+ affinity chromatography followed by cleavage of the SUMO moiety by ULP1 protease. In brief, bacterial pellets were resuspended in buffer and lysed by sonication and cellular debris removed by centrifugation. The supernatant was filtered through a 0.2-μm filter, applied to a Ni2+ affinity resin, and eluted with imidazole. The eluted protein was dialysed overnight in the presence of ULP1 protease, and subjected to Ni2+ chromatography a second time to remove the SUMO tag and ULP1 protease.

All proteins containing the NC domain were purified with additional steps for more stringent removal of nucleic acid. Following bacterial lysis and centrifugation, nucleic acid was precipitated by addition of 0.03% (v/v) polyethyleneimine followed by centrifugation. Ammonium sulfate to 20% saturation was added to the resulting supernatant, and the precipitate was resuspended in buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 2 mM TCEP (tris(2-carboxyethyl)phosphine), 5 μM ZnCl2). The protein was then purified by anion exchange and Ni2+ chromatography as above. All purification steps were performed at 4 °C or on ice. All of the final purified proteins, at concentrations of 2–5 mg/ml and having A260/A280 ratios of <0.6, were flash-frozen in liquid nitrogen and stored at –80 °C.

In vitro assembly

Assembly of s-CANC VLPs was performed by dialysing 50 μM protein against buffer (50 mM MES, pH 6 or 50 mM Tris-HCl, pH 8, 100 mM NaCl, 5 μM ZnCl2, 2 mM TCEP) with a single-stranded 50-mer oligonucleotide (GT25) at a 1:5 molar ratio of oligonucleotide to protein for 4 h at 4 °C. All reactions were adjusted to a final volume of 100 μl with buffer following dialysis. Working stocks of 10 mM inositol phosphates were made fresh (IP6, TCI cat# P0409; IP3–IP5, Cayman Chemical cat #s IP3-60960, IP4-60980, and IP5-10009851) with the pH adjusted to 6.0 with NaOH, and added both to the assembly reaction and dialysis buffer. Both s-CASP1 and CACTDSP1 assembly reactions were performed as described for s-CANC but with 500 μM protein and 500 μM IP6. Mature CA assembly was performed by dilution into buffer (50 mM MES, pH 6, 100 mM NaCl) to 250 μM final concentration in the presence of increasing amounts of IP6. Note that under these low-salt conditions, HIV-1 CA does not spontaneously assemble efficiently. The mature reactions were diluted 1:10 before spotting on EM grids. All VLP assemblies were visualized by EM negative staining with uranyl acetate. Quantification was performed by counting particles on at least five images from at least two different assembly reactions. Box plot; centre lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software24; whiskers extend to minimum and maximum values.

CRISPR knockout

The lentiCRISPR v2 vector was a gift from F. Zhang (Addgene plasmid # 52961)26. The VSV-G expression vector27 was obtained through the NIH AIDS Research and Reference Reagent Program. HEK293FT cells were purchased from Invitrogen. Cell lines were tested for, and showed no mycoplasma contamination. The plasmid v906 is an HIV-1 NL4-3 derived provirus lacking Vpr, Vif, Env, and containing CMV GFP in place of Nef. The construct has several silent restriction sites added to the CA domain of Gag for cloning purposes. The IPPK-targeted guide RNA (5′-AACAGCGCTGCGTCGTGCTG-3′) was cloned into lentiCRISPR v2, which was then used to transduce 293FT cells, followed by selection with puromycin at 1 μg/ml. Clonal isolates of the stably transduced cells were obtained by limiting dilution. To confirm the knockout, genomic DNA was isolated from clonal isolates using the DNeasy blood and tissue kit (Qiagen) following the manufacturer’s protocol. The guide RNA target sequence was amplified from genomic DNA using primers 5′-GAAATGTGTGCCACTGTGTTTA-3′ and 5′-ATGATGGACACACCACTTTCT-3′. The PCR product was directly sequenced.

Infectivity assays

Equivalent numbers of 293FT WT or IPPK KO cells were plated in 35-mm dishes and transfected with 900 ng of v906 and 100 ng of VSV-G. Medium was collected two days post-transfection and frozen at −80 °C to lyse cells in the supernatant. Thawed supernatants were centrifuged at 1,500g for 5 min to remove cellular debris. Infections were performed in fresh 293FT cells. Cells were collected two days later, fixed with 4% paraformaldehyde, and analysed for GFP expression using an Accuri C6 flow cytometer.

Two-dimensional crystallography

CACTDSP1 2D crystals were produced by incubating 0.8 mM protein with 0.8 mM IP6 at room temperature for 30 min. Samples were placed on a carbon-coated grid, washed with 0.1 M KCl, blotted to near dryness and flash frozen by plunging in liquid ethane. Low-dose images were collected on a Tecnai F20 equipped with 4k × 4k Ultrascan CCD camera (Gatan) under low electron-dose conditions (~20 e−/A2). Images were converted to MRC format and manual indexing, unbending, and corrections for CTF were performed with 2dx28.

X-ray crystallography

Purified CACTDSP1 protein (stock = 4.5 mg/ml) was mixed with equal volume of IP6 (stock = 1.4 mM) and incubated briefly at room temperature. Crystals were formed by the vapour diffusion method in sitting drops containing a 1:1 ratio of the protein/IP6 mix and precipitant (0.2 M NaCl, 20% PEG 3,350, 0.1 M Bis-Tris, pH 5.35). Hexagonal plate crystals grew after 2 days of incubation at 17 °C. Crystals were cryoprotected in 25% ethylene glycol and flash-frozen in liquid nitrogen. Diffraction data were collected at the Advanced Photon Source beamline 22-ID and were indexed and scaled with HKL200029. The structure was solved by molecular replacement using as search model one monomer of the previously reported CACTDSP1 hexamer structure (PDB 5I4T)3, with Lys290 and Lys359 sidechains truncated at Cα. Refinement and model building was performed using the PHENIX suite of programs30 and Coot31. Refinement of the protein was first completed before modelling the IP6 and Lys290/Lys359 sidechain densities. The IP6 density was unambiguously identified from mFo−DFc difference maps, and the interpretation that the density was due to bound IP6 was further supported by comparison with difference maps from our previously reported CACTDSP1 structure in the absence of IP63. Given the resolution of the data and crystallographic averaging of the ligand density, we assumed that the bound IP6 was in the myo conformation and refined the ligand as a rigid body with 1/6 occupancy. Only weak residual difference densities were observed after this treatment, suggesting that the modelled IP6 conformation was a reasonable interpretation of the data.

Disulfide-stabilized CA A14C/E45C/W184A/M185A was prepared as previously described32,33. IP6-containing samples were prepared for crystallization as described for CACTDSP1, except that the protein stock concentration in this case was 10 mg/ml. P6 crystals were obtained in precipitant containing 2% Tacsimate, 14% PEG 8,000, 0.1 M Tris, pH 8.4, whereas P212121 crystals were obtained in 8% PEG 8,000, 0.1 M Tris, pH 8.2. Data were collected at Advanced Photon Source beamline 22-BM (P6 form) or 22-ID (P212121 form) and processed with HKL200029. The crystals were isomorphous with previously deposited structures solved in the absence of IP6 (PDB 3H47 and 3H4E)32, and so initial refinement was through rigid body placement of the deposited coordinates (with Arg150 sidechains and waters removed). Refinement of protein-only models were first completed before modelling the IP6 and Arg150 sidechain densities. As with the immature hexamer, IP6 densities were unambiguously identified by mFo−DFc difference maps and by difference density comparisons of CA hexamers crystallized with and without IP6. The IP6 densities in the mature hexamers were modelled as follows. For the P6 crystal form, a single well-defined IP6 density was found inside the β-hairpin chamber (Fig. 3c–e). As in the case of the immature hexamer, the ligand density was also six-fold symmetric due to crystallographic averaging, but in this case indicated at least two binding modes, one with the axial phosphate pointing away from the Arg18 ring and a second pointing towards the ring. Two IP6 molecules were therefore docked into the density, again in the myo form and refined as rigid bodies with 1/12 occupancy (Fig. 3e). Again, only weak residual difference densities were observed after this treatment, suggesting that the modelled IP6 conformations were reasonable interpretations of the data. For the P212121 form, IP6 densities were observed on both sides of the Arg18 ring (there were 2 hexamers in the asymmetric unit and so we observed 4 density features) (Extended Data Fig. 8). These were modelled by docking myo-IP6 in one or two configurations that appeared most consistent with the local density distribution, and then refined as rigid bodies with appropriate occupancy. In this case, significant residual difference densities were observed at the ligand positions after refinement, indicating that additional binding modes were possible. However, we did not attempt to model these multiple overlapping binding modes. The P6 form crystallized in the presence of Tacsimate (Hampton Scientific), which is a mixture of organic carboxylic acids. The excess of negatively charged precipitant therefore appears to have inhibited binding of IP6 below the Arg18 ring; this can be reasonably interpreted to mean that IP6 has greater affinity for the site enclosed by the β-hairpins. The P212121 form crystallized in the absence of Tacsimate, allowing IP6 binding on both sides of the Arg18 ring.

Statistics for all three crystal structures are reported in Extended Data Table 1. Structure visualizations and images were made by using PyMol (Schrödinger Scientific).

Molecular dynamics simulations

The structure of the IP6-bound CACTDSP1 hexamer was used to derive bound and unbound CACTDSP1 models. The IPs and mellitic acid molecules were placed in the central pore of the hexamer between K290 and K359 rings in the corresponding models by aligning the carbons present in the central cyclic ring. All models were then solvated with TIP3P water34 and the salt concentration was set to 150 mM NaCl. Sixteen chloride molecules and twenty-three sodium ions were placed near the hexamer using the CIONIZE plugin in VMD35 to minimize the electrostatic potential. The resulting CACTDSP1 models each contained a total of 30,000 atoms.

After model building, the systems were initially subjected to minimization in two stages, both using the conjugated gradient algorithm36 with linear searching37. Each stage consisted of 10,000 steps of energy minimization. During the first stage, only water molecules and ions were free to move, while the protein and IP molecules, if any, were fixed. In the second stage, the backbone atoms of the protein were restrained with a force constant of 10.0 Kcal mol−1 Å−2 Convergence of the minimizations were confirmed once the variances of gradients were not greater than 1 Kcal mol−1 Å−1. During thermalization the systems were heated from 50 K to 310 K in 20 K increments over 1 ns. Subsequently, the systems were equilibrated, while the backbone atoms of CACTDSP1 were restrained. The positional restraints were gradually released at a rate of 1.0 Kcal mol−1 Å−2 per 400 ps from 10.0 to 0.0 Kcal mol−1 Å−2. NAMD 2.1238 was employed during minimization/thermalization and equilibration steps.

Simulations of IP6-bound and unbound CACTDSP1 were then performed on the special purpose computer Anton239 in the Pittsburgh supercomputing centre for 2 μs. The CHARMM 36m40 force-field was used for all simulations. Parameters for IP6 were derived by analogy following the CGENFF protocol41. During the simulation, the temperature (310 K) and pressure (1 atm) were maintained by using the Multigrator integrator42 and the simulation time-step was set to 2.5 fs/step, with short-range forces evaluated at every time step, and long-range electrostatics evaluated at every second time step. Short-range non-bonded interactions were cut off at 17 Å; long range electrostatics were calculated using the k-Gaussian Split Ewald method43.

Simulations of CACTDSP1 bound to IP3, IP4, and IP5 were performed for 2 μs on TACC Stampede 2 using NAMD 2.1238. The molecular simulations were conducted under isothermal (310 K) and isobaric (1 atm) conditions, regulated by the Langevin thermostat44 and the Nosé-Hoover Langevin piston45,46, respectively. All bonds to hydrogen atoms were constrained with the SHAKE algorithm47. A time step of 2 fs was used for all simulations. Long-range electrostatics were calculated using the Particle-Mesh-Ewald method, as implemented in NAMD38, with a cutoff of 1.2 nm. Full electrostatic interactions were calculated every two time steps while nonbonded interactions were performed every time step.

Analysis of MD simulations

Root-mean-square deviations (RMSDs) and root mean square fluctuations (RMSFs) of the Cα of the CACTDSP1 hexamers were computed using the measure command in VMD35. Before RMSD and RMSF calculations, the structure of the hexamer was aligned to a common reference. RMSFs of each monomer in a central hexamer were calculated to obtain RMSF standard deviations of an entire hexamer.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Coordinates and structure factors have been deposited at the RCSB Protein Data Bank (PDB) database, under accession numbers 6BHR, 6BHT, and 6BHS. All other data are available from the authors on request; see author contributions for specific datasets.