DENND5B variants

DENND5B (DENN domain-containing protein 5B) is a GTP exchange factor (GEF) for RAB39A and RAB39B.

• Proline-237 (found mutated to serine) in the cDENN domain. Prolines have different backbones to other amino acids and are mutations in short loops are generally not tolerated. Predictions on the AlphaFold2 model suggest it may be destabilising (6.2 kcal/mol).
• Serine-559 (found mutated to leucine) in the dDENN domain. Surface mutation facing and in close promixity to the RUN domain —within 5Å of threonine-776, which is the length of a water bridge.
• Arginine-749 (found mutated to histidine) in the 1st RUN domain. Surface mutation facing the dDENN. Potentially pathogenic via the same mechanism as S559L.
• Serine-800 (found mutated to leucine) in the 1st RUN domain. Last residue in a turn and forms a hydrogen bond with E786 in the previous helix via its side chain, which would be lost in a leucine variant. In fact, Predictions suggest it to be highly destabilising (>10 kcal/mol).
• Aspartate-849 (found mutated to glutamate) in the 1st RUN domain. This buried charged residues forms salt bridges with lysine-876 and histine-882. Despite the similarity of the two residues, glutamate is longer and predictions suggest it to be highly destabilising (6 kcal/mol).
• Histidine-852 (found mutated to phenylalanine) in the 1st RUN domain. This buried residue is on the following rung of the helix as D849 and its sidechain is appears packed in two alternative conformations depending on the distance to β-hairpin 1015-1019 of the PLAT/LH2 (which shifts by 1 Å). Both conformations form several hydrophobic interactions, but in the more distant conformation (shown), it forms π-π interactions with tryptophan-927 and a π-sulfur interaction with methionine-856, while in the other it forms a hydrogen-bond with the backbone of a preceeding residue. If this actually represents two biologically relevant states, any change would disrupt this balance.
• Arginine-904 (found mutated to histidine) in the PLAT/LH2 domain. This charged residue is found in close proximity to several other charged residues (Lys894, Arg899, Arg909) and less than 10 Å away from where the farnesyl-anchor of Rab39A/B would be. Given that this domain family is known to be membrane bound, and clusters of positively charged surface residues in such protein bind phospholipid, this residue may play a role in anchoring the protein in the membrane where it interacts with Rab39A/B.

The protein can subdived into the following domains:

• uDENN, upstream part of the DENN tripartite domain
• cDENN, central part of the DENN tripartite domain
• dDENN, downstream part of the DENN tripartite domain
• first RUN, a domain involved in signalling
• PLAT/LH2, a membrane or protein interaction domain
• second RUN domain

### Model

ColabFold generated model (AF2 multimer v2, 48 recycles) with the GTP and Mg taken from PDB:3CWZ (73% sequence identity, R6IP1-RAB6).