Poster Presentation Crystal36-AXAA Conference 2026

Structural characterisation of INPP4B: insights into the final unsolved human phosphoinositide phosphatase family (147841)

Gareth Jones 1 , Charles Bayly-Jones 1 , Hariprasad Venugopal 2 , Chris Lupton 1 , Andrew Ellisdon 2
  1. Biomedicine discovery institute , Monash University, Melbourne, VIC, Australia
  2. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, Victoria, Australia

Phosphoinositides (PI) are a minor component of the cellular membranes, however, they play a crucial role in cell biology as signalling molecules. The specific function of a PI is dictated by the phosphorylation state of its inositol headgroup, which can be variably and combinatorially phosphorylated at the 3ˈ-, 4ˈ-, and 5ˈ- positions. The phosphorylation state is controlled by a plethora of kinases, which attach phosphate groups, and phosphatases, which remove phosphate groups. This thesis will focus on the PI- phosphatases, of which there are ~ 38 encoded within the human genome. These phosphatases can be divided into four groups based on their positional specificity. These groups are the 3ˈ-, 4ˈ-, 5ˈ-, and Sac domain PI-phosphatases. The first three families hydrolyse 3ˈ-, 4ˈ-, and 5ˈ-phosphates, respectively, while the Sac domain phosphatases exhibit broader substrate specificity. Structural representatives of the 3ˈ-, 5ˈ-, and Sac domain phosphatases have been solved, and the molecular basis of these proteins’ specific activities is well understood [1, 2, 3]. However, to date no structure of an active human 4ˈ-PI-phosphatase has been solved. Consequently, the molecular basis of 4ˈ-PI-phosphatase activity is comparatively poorly understood.

Here-in, I use cryo-electron microscopy (cryo-EM) to solve the structure of apo-INPP4B to a resolution of 3.2 Å. This is the first structure of an active human 4ˈ-PI-phosphatase and only the second structure of a 4ˈ-PI-phosphatase from any species. The structure reveals a typical DUSP catalytic unit embedded within a novel protein scaffold that bears little resemblance to other human PI-phosphatases. Comparative analysis with bacterial PI-phosphatase structures enabled the identification of structural features surrounding the INPP4B active site that may impart 4ˈ-PIphosphatase activity. These observations were then validated, using the apo-INPP4B structure and AI-based protein modelling guided extensive functional mutagenesis experiments. These experiments suggest that INPP4B operates via a typical DUSP catalytic mechanism and that the unique context of the IP4P scaffold imparts the specificity of INPP4B activity.

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  3. Tsujishita Y, Guo S, Stolz LE, York JD, Hurley JH. (2001) Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase. Cell.105(3):379-89 https://doi.org.10.1016/s0092-8674(01)00326-9