H323R

H323 is held between His322 (2.45 Angstrom) and Ile324 (2.46 Angstrom) covalently. Through-space neighbors read Pro320 (3.53 Angstrom), Ala326 (4.25 Angstrom), Thr321 (4.49 Angstrom), Asn325 (4.49 Angstrom), and Leu327 (4.93 Angstrom). The pair of adjacent histidines at 322-323 is structurally distinctive: two imidazole side chains separated by a single peptide bond, both within a transmembrane helix. The wild-type arrangement likely places both imidazoles facing inward toward each other or coordinating with the polar Thr321 and Asn325, forming a small polar cluster within the helix interior.

Replacing H323 with arginine introduces a fully charged guanidinium where a partial-charge imidazole sat. The thermodynamic problem is desolvation: arginine in a bilayer interior carries a desolvation cost of 8-12 kcal/mol if fully buried, less if its side chain can snorkel toward an interfacial water network. The presence of nearby polar residues (Thr321, Asn325, the H322 imidazole) provides some partial solvation, but the local geometry was not built to host a permanently charged residue.

The structural consequence is one of two outcomes: either Arg323's guanidinium snorkels toward the cytoplasmic face of TM1 (potentially dragging the helix slightly out of register), or it remains buried at substantial energetic cost. Either outcome perturbs TM1 packing against its neighboring helices.

DynaMut2's DeltaDeltaG of -0.02 kcal/mol — essentially zero — is a known weakness of energy functions for buried charge substitutions. The function captures local van der Waals and hydrogen-bond changes but under-weights desolvation. AlphaMissense at 0.687 is closer to a true read of the disruption: clearly pathogenic (above the 0.564 threshold) but not the highest-scoring variant in this batch, consistent with the imidazole-to-guanidinium swap being chemically less drastic than e.g. a glycine-to-arginine substitution.