ITPR3

Inositol 1,4,5-trisphosphate receptor type 3 (Uniprot: Q14573) is a calcium channel activated by inositol 1,4,5-trisphosphate (I3P). It forms a homotetramer on the ER membrane. At the N-terminal, there is a large cytosolic domain . Within the cytosolic domain, I3P is bound. The bulk of the cytoplasmic region is composed of helix bundles protruding outwards, but the initial residues (far from the membrane) form two consecutive small beta-barrels, the first, referred to as the coupling domain binds to part of a helical bundle domain from another monomer —both in the cytoplasmic side. In the cryoEM structures the region where the coupling domain binds differs depending on the presence of calcium and IP₃. The presence of IP₃ in the cytosol forced an altered conformation making the channels more permeable to calcium ions from the ER. This flux is further increased by the precence of calcium in the cytosol.

The C-terminal transmembrane region (2145-2537) features two narrowings in the pore, one being the gate of the channel, formed by I2517 and F2513, which regulates the flow of calcium across it and the other the selectiving filter formed by N2472 and D2478.

Variants

A196T

• wild type
• mutant
• overlay

This mutation is in the N-terminal coupling domain.

The mutation goes from a smaller hydrophobic residue to a larger polar one in the core of the domain, which forces it and nearby residues outwards, including K208. In light of different binding modes with the other monomer in the presence of IP₃ and calcium this mutation has different effects, the open form is stabilised, while the closed forms, especially the calcium inhibited form, are destabilised (+4.9 kcal/mol in the high calcium and IP₃ model 6DRC), meaning that an open pore state is likely more favourable.

I2506N

• wild type
• all four wild type
• mutant
• all four mutant
• overlay
• See also surface view to gauge the channel (NB. wait 2–3 seconds as its computationally intensitive on the browser. Use Control+Shift+Wheel to change clipping).

The mutation replaces a hydropholic residue for a polar one on the walls of the channel near the the channel gate and N2472 and D2478 (combined). When the pore opens the helices it is on, adopt a π bulge centred around I2507. Therefore, its location makes it fully reasonable that it may interfere with the dynamics of the protein and the passage of calcium. This is conformed by in silico calculation, where the open state is mildly stabilised (-1.3 kcal/mol), while other conformations are not. The residues from different chains are 12 and 17 Å away so are likely to influence each other as the calculated ∆∆G shows some epistasis.

R2524H and R2524C

• wild type (docked)
• all four wild type
• R2524H mutant
• all four R2524H mutant
• R2524H overlay
• R2524C mutant
• all four R2524C mutant
• R2524C overlay

These are mutations at the interface of the cytosol and plasma membrane and flank the gating residues. The mutations are deleterious for the closed unbound state (R2524H: +2 kcal/mol; R2524C: +2 kcal/mol), but less so in the open state (R2524H: -2.7 kcal/mol; R2524C: 1.1 kcal/mol) which is likely to affect the permeability of the channel. The destabilisation is due to the fact that arginine 2524 forms two salt bridges (generally, a salt bridge contributes –2 kcal/mol), while the smaller histidine can form only one hydrogen bond, while cysteine none (as reflected in the worse value of the Columbic electrostatic forces term, fa_elec), but the tilt in the π bulge conformation, moves these groups away.

A minor potential observation involves the fact that arginines commonly form positively charged pockets at the interface in order to bind the phosphate heads of phospholipids. In some cases it controls localisation across the membrane by exploiting the difference in composition of lipid rafts. The electron density map show something in the pocket, most likely a water, but it may natively bind a phospholipid —stripped in sample preparation. In the offchance a model with an organophosphate docked in this pocket (K2527, R2524, N2357) was made. In this model, the binding of the organophosphate is also weakened in R2524H (wild type/ mutant/ overlay, +2.8 kcal/mol) and R2524C (wild type/ mutant/ overlay,+2.4 kcal/mol).

V615M

• wild type
• V615M mutant
• V615M overlay

This variant reported by Ronkko et al. 2020 is on the hinge of the conformational change in the helical bundle domain near the IP3 binding pocket and disfavours certain conformations more than others (e.g. in the unbound form +2.4 kcal/mol difference), while favours the active and inhibited forms due to different contacts in the neighbourhood.

ITPR1 and Gillespie Syndrome

In ITPR1, G2554R, K2611∆, E2109G (canonical ITPR1 numbering) cause Gillespie Syndrome in a dominant fashion (McEntagart et al. 2016). The equivalent positions in ITPR3 of these are:

E2005G

• wild type
• G2473R mutant
• G2473R overlay

This mutation in the transmembrane domain is destabilising (4.2 kcal/mol).

K2530∆

• wild type

This mutation is on a helix above the transmembrane domain so it is bound to change the structure. However, it is water exposed so won’t make the protein unfolded, but just twist the helix and change its behaviour.

E2005G

• wild type
• E2005G mutant
• E2005G overlay

This is above transmembrane region in the core of a helix bundle formed by different parts of the protein, not overall destabilising, but does change the balance of forces most likely making more mobile.

gnomAD

There are 19 homozygous variants in gnomAD 3 control dataset. 7, including an in-frame deletion, are in missing density regions (so unlikely to be pathogenic). Three mutations are on the channel domain but most likely tolerated:

• V2295M (wild type/ mutant/ overlay) is membrane facing but conserves the hydrophobic character (neutral)
• I2273T (wild type/ mutant/ overlay) is at the membrane surface away from the channel (not destabilising)
• E2398Q (wild type/ mutant/ overlay) is on the surface of the wide lumenal vestibule (neutral and rather conservative)

One mutatation has a predicted ∆∆G greater than 2 kcal/mol, but barely. Namely, L374W (wild type/ mutant/ overlay) is on a large loop and is slightly destabilising (+2.1 kcal/mol, caused by unfavourable sidechain torsion angle, not solvatation)

Of interest is P187S (wild type/ mutant/ overlay) which is on the interface beta-barrel as A196. This mutation is predicted to be destabilise all conformations equally, without favouring the open pore state as seen for A197T.

This further indicates that there is no evidence for a recessive complete loss of function of this protein in the healthy population, consistent withe pLI of the gene.

Truncations appear in gnomAD. Due to the C-terminal location of the transmembrane domain, a truncation cannot not partake in tetramer assembly. This rules out haploinsufficiency as a mechanism of pathogenicity and implies that if a mutant chain partakes in the tetramer, it would not be pathogenic if the tetramer required two or more mutant chains to be non-functional —this conclusion does not include the case where a single chain renders the whole tetramer non-functional (accounting for 15/16 cases).

Of the 97 heterozygous variants in gnomAD more frequent than 5e-5, six are deleterious:

• Y2054C (wild type/ mutant/ overlay, +4.5 kcal/mol)
• L2114R (wild type/ mutant/ overlay, +3.8 kcal/mol)
• R2262H (wild type/ mutant/ overlay, +3.5 kcal/mol)
• F977L (wild type/ mutant/ overlay, +7.0 kcal/mol)
• V1356M (wild type/ mutant/ overlay, +2.3 kcal/mol)
• V1058M (wild type/ mutant/ overlay, +2.1 kcal/mol)

However, of the 19 less frequent variants that fall within 8 Å of a ligand or the interface, six stand out. Namely,

• D2582H (wild type/ mutant/ overlay) affects calcium binding,
• R270H (wild type/ mutant/ overlay) affects IP₃ binding,
• R2471H (wild type/ mutant/ overlay) disrupts the chain interface at the lumenal vestibule (R2524 is on the cytosolic side, close to the gate) ₃ (2.3 kcal/mol complex, 1.2 kcal/mol interface)
• G2477A (wild type/ mutant/ overlay) does not weaken the overall stability but weakens the interface (+1.7 kcal/mol) at the lumenal side.
• L2511V (wild type/ mutant/ overlay) is close to the gate and weakens the complex by 1.2 kcal/mol. This is less than the nearby I2506N and R2524H/C but it is very salient as these are farther away
• I184S (wild type/mutant/overlay) on the coupling domain destabilises the domain by 1.8 kcal/mol but does not affect interface binding.

Given the fact that some of have a destabilising effect similar to the pathogenic variants, it suggests that the pathogenic variants are unlikely to be pathogenic by resulting in loss of activity in all 15/16 configurations of the tetramer (else R2471H would be also pathogenic for example), but may result in deregulation of the tetramer. The presence of R270H and D2582H hints that greater activation threshold may also be tollerated somewhat. If the pathogenic variants were to result in a more leaky resting state as a result of a less stable gate conformation, the effect of L2511V need to be addressed.

Methods

Initial model show: PDB:6DRC. (*) NB: the models have been cutdown and reoriented for better and faster viewing. Also the total score reported for R2524H and R2524C are based on 6DRC, but the links show the ethylphosphate bound derivative reducing the number of models to load. Data, method description and code in github repo