Fig 2: Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) defines nanobody 91 binding sites with PRL-3.(A) PRL-3 in complex with nanobody 91 shows regions of increased (red) and decreased (blue) deuterium uptake, compared to apo-PRL-3. Heatmap indicates approximately 70% sequence coverage by mass spectrometry; gray areas represent portions of PRL-3 where data for deuterium exchange was not recovered. (B) Peptides 13–19 on PRL-3 were deprotected following nanobody binding, while peptides 56–79 and 132–146 showed decreases in deuterium uptake, reflecting more protection by nanobody 91 on PRL-3 in these regions.

Fig 3: Nanobodies selectively immunoprecipitate PRL-3 from HEK293T cell lysate and IP HA-PRL-3 comparatively to commercially available anti-PRL-3 antibodies.(A) Nanobody 19 pulls down 3XFLAG-PRL-3 with minimal to no pulldown of 3XFLAG-PRL-1 or 3XFLAG-PRL-2. Successful nanobody coupling to Dynabeads was verified using an antibody against the C-terminal 6XHis-tag present on each nanobody. (B) Nanobody 19 pulls down HA-PRL-3 from HCT116 cells just as well as commercially available antibodies MAB3219 and sc-130355, and better than ab50276 and GTX100600. The input, transiently overexpressed HA-PRL-3, is shown separately. Nanobody 19 was coupled to superparamagnetic Dynabeads® M-270 Epoxy beads and used in immunoprecipitation assays with lysates from HEK293T cells transduced with 3XFLAG-PRL-1, -2 or -3 and HCT116 cells transiently transfected with HA-PRL-3. All anti-PRL-3 antibodies were incubated with HA-PRL-3 lysate and pulled down using Protein A and Protein G beads.

Fig 4: PRL-3 has numerous predicted metal binding sites. The crystal structure of PRL-3 in complex with the CBS domain of CNNM3 (PDB:5TSR) was analyzed using the metal ion-binding site prediction and modeling server (MIB2). The CBS domain (yellow) inserts a protruding loop into the active site of PRL-3 (gray). Predicted calcium, magnesium, manganese, and zinc binding sites are shown in orange, blue, green, and pink, respectively, with the PRL-3 catalytic site in red. Several predictions place metal ions directly in the CNNM interface or interacting with the nearby active site and WPD loop of PRL-3.

Fig 5: Divalent metals do not alter the formation or hydrolysis of the PRL-3 phospho-cysteine intermediate. (A) Recombinant PRL-3 was incubated with DiFMUP and the metal salts for the designated time period. The samples were then analyzed using Phos-Tag acrylamide and gel electrophoresis to separate the non-phosphorylated PRL-3 (bottom bands) from the phospho-cysteine intermediate form (top bands). Cysteine phosphorylation is sensitive to boiling, as demonstrated in the last lane as a control. (B) Band quantifications of each condition to estimate the percent of PRL-3 in the phospho-cysteine intermediate state normalized to the no DiFMUP control in lane 1.