Journal: BBA Advances

Article Title: Claudin-11, a hypomyelinating leukodystrophy 22 (HLD22)-responsible protein, uniquely interacts with shroom-2 to change cell phenotypes

doi: 10.1016/j.bbadva.2025.100159

Figure Lengend Snippet: Key materials used in this study.

Article Snippet: pcDNA3.1(+)-N-DYK-human Shroom2 PDZ domain (1–100 amino acids) , Genscript , J377 × 912G0–6 , R633123 , 1.25 μg of DNA per 6 cm dish.

Techniques: Western Blot, Sequencing, Immunoprecipitation, Concentration Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Mental health disorder-associated missense mutations within human RGS12 and its conserved architecture among mammalian species. ( A ) Locations of rare missense mutations associated with attention-deficit/hyperactivity disorder (ADHD), bipolar disorder (BD), or schizophrenia/schizoaffective disorder (SCZ) within the multi-domain architecture of the human RGS12 protein. Multi-domain architecture map of the human RGS12 protein (UniProt O14924, a.k.a. RGS12_HUMAN [1447 aa]) has been placed above an x -axis scale of amino acids. Known functional domains within RGS12 include a PDZ (PSD-95/discs-large/ZO-homology) domain, a PTB (phosphotyrosine-binding) domain, an RGS-box (“Regulator of G protein Signaling” domain), a tandem repeat of RBD (Ras-binding) domains, and a GoLoco (Gα i/o -locomotion defects) motif; pink regions represent low-complexity polypeptide sequences predicted to lack secondary or higher-order structure. The R59Q missense mutation detected in BD-affected family members is seen to be in the middle of the PDZ domain sequence (highlighted in red). ( B ) Conservation of the multi-domain architecture of RGS12 amongst the orthologues of 74 other mammalian species. Phylogenetic tree of 75 mammalian RGS12 orthologues, represented by respective species’ names and grouped into clades with other mammalian species that share similar evolutionary origins. The protein domain architecture of each RGS12 orthologue is portrayed adjacent to their species nomenclature and consists of the same domain symbols as shown in panel A and derived from the Simple Modular Architecture Research Tool (SMART) database. The RGS12 PDZ domains of four Australian marsupials (names in orange) that contain a lysine rather than an arginine at the position characterized by the human R59Q missense variation are highlighted with yellow asterisks (*) within the orange hexagons that represent PDZ domains.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Functional Assay, Binding Assay, Mutagenesis, Sequencing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: ( A ) Sequence and secondary structure similarity between human RGS12 PDZ domain (top row; aa 30–99 of UniProt O14924) and human NHERF1 PDZ1 domain (bottom row; aa 22–94 of UniProt O14745): −, gap; +, similar amino acid; α, alpha-helical character; β, beta-strand character. Secondary structure derived from PDB IDs 2KV8 and 4JL7, respectively. ( B , C ) 3D and 2D representations of the NHERF1 PDZ1 domain/CXCR2 C-tail interaction (“TSTTL-COOH”), rendered via Maestro using PDB id 4JL7. Hydrogen bonds between the PDZ domain and bound pentameric ligand are depicted as yellow dotted lines in panel ( B ) and purple arrows in panel ( C ).

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Sequencing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Heat map of GLIDE SP-peptide and GLIDE XP scores for predicted docking of four pentameric ligands into the ligand-binding “receptor grid” specified for each of the 20 low-energy structural models (“poses”) of the human RGS12 PDZ domain recorded within PDB id 2KV8. GLIDE SPpep and XP scores are indexed similarly to Gibbs’ free energy but do not explicitly use kcal/mol units; a more negative value indicates that tighter binding is predicted. Ligands used in docking include the CXCR2 C-tail TSTTL-COOH, the MEK2 C-tail TRTAV-COOH, the SAPAP3 C-tail AQTRL-COOH, and a negative control derived from the C-terminus of the mouse Notch1 protein (PEAFK-COOH) previously established to have no demonstrable binding to recombinant RGS12 PDZ protein [ 19 , 42 , 65 ]. Note that pose 16 provides the darkest green shading for each GLIDE score, except for the negative control peptide.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Ligand Binding Assay, Binding Assay, Negative Control, Derivative Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Structural models of the RGS12 PDZ domain/CXCR2 C-tail complex, as derived from GLIDE XP (panels A , B ) and GLIDE SPpep (panel C ) docking algorithms. ( A , B ) Three- and two-dimensional representations, respectively, of the predicted RGS12 PDZ domain/CXCR2 C-tail complex, rendered via Schrödinger’s Maestro after GLIDE XP docking of the free TSTTL-COOH pentameric peptide into the structural coordinates of low-energy pose 16 of PDB id 2KV8. Dotted black lines in panel A represent the distances measured in the 3D model between a hydrogen of the arginine-59 side chain and an oxygen of a potential hydrogen-bonded neighbor (asparagine-54; 2.2 Å) and between the center of the Arg59 side chain’s guanidinium group and the central carbon of the TSTTL-COOH ligand’s carboxylic acid moiety (17.8 Å). ( C ) Two-dimensional representation of the predicted RGS12 PDZ domain/CXCR2 C-tail complex, rendered after GLIDE SPpep docking of the free TSTTL-COOH pentameric peptide into the structural coordinates of low-energy pose 16 of PDB id 2KV8.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Tracking predicted distances of Arg59 from neighboring Asp54 and the G-Φ-G-Φ motif by MD simulations. ( A ) Depictions of the solvent-accessible arginine-59 (light blue) and asparagine-54 (orange) side chains at the start (time “zero”) of an MD simulation of the unliganded RGS12 PDZ domain (pose 16, PDB id 2KV8) and after 1.0 nanoseconds have elapsed. Plotted underneath is the predicted distance (over 200 ns of MD simulation) between the nearest nitrogen of the arginine-59’s guanidium side chain and the carbonyl–oxygen of the neighboring asparagine-54 side chain within pose 16 (magenta line) and separately for pose 3 (dark blue line). Dotted line indicates average distance over 200 ns of 11.4 ± 1.5 Å (mean ± s.d) for pose 16. ( B ) Depiction of the solvent-accessible arginine-59 and glycine-33 (green; the second glycine of the G-Φ-G-Φ motif) at the start (time “zero”) of an MD simulation of the unliganded RGS12 PDZ domain (empty ligand-binding cavity highlighted in an electrostatics-shaded surface: blue = electropositive and red = electronegative). Plotted to the right is the predicted distance (over 200 ns) between the central carbon (CZ) of the arginine-59’s guanidinium side chain and the alpha-carbon (CA) of glycine-33 for pose 16 (green line) and pose 3 (brown line); dotted line indicates average distance over 200 ns of 15.9 ± 0.6 Å (mean ± s.d) for pose 16. ( C ) Average distances over 200 ns MD trajectories obtained for both residue pairs in separate simulations of other poses of the RGS12 PDZ domain from PDB id 2KV8.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Solvent, Ligand Binding Assay, Residue

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Model of SAPAP3 bound to the RGS12 PDZ domain and in vitro evidence of interaction. ( A ) Three-dimensional electrostatics-shaded surface representation (blue = electropositive, red = electronegative) of the 16th low-energy pose of RGS12 PDZ domain (PDB id 2KV8), as bound by the AQTRL-COOH pentamer that represents the C-terminal tail of the SAPAP3 protein (hydrogen bonds indicated by dashed yellow lines). ( B ) Two-dimensional map, derived from panel A, of hydrogen bond interactions (purple arrows) between the AQTRL-COOH pentamer and RGS12 PDZ domain residues, as predicted by in silico docking (GLIDE XP score −12.1). ( C ) Reciprocal co-immuno-precipitation of HA-epitope tagged RGS12 and Flag-epitope tagged SAPAP3 exogenously expressed in HEK293T cells, confirming previously published observations of an RGS12/SAPAP3 interaction from a yeast two-hybrid screen with N-terminal RGS12 PDZ and PTB domains as the bait molecule and a mouse whole brain cDNA library as the prey . IB, immuno-blotting; IP, immuno-precipitation; no Ab, no primary antibody control; αHA, anti-HA epitope primary antibody; αFLAG, anti-Flag epitope primary antibody; Lysates, whole-cell lysates separated by SDS-PAGE to control for expression and gel-loading; kDa, kiloDaltons (positions of co-electrophoresed marker proteins). ( D ) The PDZ domain of RGS12 binds directly to the C-terminal tail of SAPAP3 in vitro. Surface plasmon resonance (SPR) was performed using separate biosensor surfaces of immobilized, biotinylated peptides from the C-terminal tails of wildtype (wt) SAPAP3 (AQTRL-COOH), a point mutant and capped version of the SAPAP3 C-tail (T964S plus amidated carboxy-terminus; “mt”), or CXCR2 C-tail (TSTTL-COOH) as a positive control for binding . Recombinant RGS12 PDZ domain protein (300 μM; ref. ) was used as the analyte. Inset: increasing concentration of RGS12 PDZ domain protein was used as the analyte to determine maximal binding to the wildtype SAPAP3 and CXCR2 C-tail surfaces.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: In Vitro, Derivative Assay, In Silico, Immunoprecipitation, FLAG-tag, Two Hybrid Screening, cDNA Library Assay, Control, SDS Page, Expressing, Marker, SPR Assay, Mutagenesis, Positive Control, Binding Assay, Recombinant, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Comparison of best docking scores and MD calculated free-energy changes for indicated RGS12 PDZ domain–peptide complexes.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Comparison, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Representative solvation states and results of MD simulations of liganded RGS12 PDZ domain point mutant variants. ( A ) Three-dimensional electrostatic surface representation (blue = electropositive, red = electronegative) of the empty ligand-binding cavity of the 16th low-energy pose of RGS12 PDZ domain (PDB id 2KV8) with an arginine-59-to-glutamine substitution; solvation with 0.15 M NaCl is illustrated in CPK coloring about the glutamine-59 position (Gln59 highlighted in green spheres). ( B ) Three-dimensional electrostatic surface representation (blue = electropositive, red = electronegative) of the ligand-binding cavity of the 16th low-energy pose of RGS12 PDZ domain (PDB id 2KV8) bound to the TSTTL-COOH pentamer (purple spheres) and containing a glycine-31-to-proline substitution (Pro31 highlighted in green spheres); solvation with 0.15 M NaCl is illustrated in CPK coloring about the bound pentameric ligand. ( C ) MD results of predicted free-energy changes (in kcal/mol; values are mean ± std. dev.) to domain folding and to indicated ligand binding using Schrodinger’s FEP Protein Mutation for Ligand Selectivity with default settings. Predictions of destabilization colored in red, and predictions of stabilization colored in green.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Mutagenesis, Ligand Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Ligand-binding affinities of R59Q-variant and wildtype RGS12 PDZ domain proteins, as measured by SPR biosensors. ( A , C ) Real-time measurements of optical response units (RUs) over time (in seconds) upon passing sequential drops of indicated concentrations of R59Q-variant RGS12 PDZ domain GST-fusion protein (panel A ) or wildtype RGS12 PDZ domain GST-fusion protein (panel C ) over streptavidin–gold nanosensors pre-bound with a negative control biotinylated peptide (mNotch1 C-tail; light blue) or biotinylated CXCR2 C-tail peptide TSTTL-COOH (black). ( B , D ) Test-surface SPR signal traces in panels ( A ) and ( C ), respectively, were subtracted from negative control surface traces to generate panels ( B ) and ( D ) prior to calculations of maximal binding (R max ), apparent on-rate (k a ), apparent off-rate (k d ), and estimated dissociation constant (K D ). Black line represents plot of idealized trace using 1:1 Langmuir kinetics model with mass transport limitation (Alto software version 2.3.1; Nicoya). ( D inset), Plots of mean ± std. dev. for maximal response (R max in RUs, circles; left y -axis) and for dissociation constant (K D in molar, diamonds; right y -axis); N = 4 for both protein analytes interacting with CXCR2 TSTTL-COOH biosensor (R59Q variant in gray; wildtype in black). ( E , F ) Control surface-subtracted SPR signal traces for R59Q-variant RGS12 PDZ domain GST-fusion protein (panel E ) or wildtype RGS12 PDZ domain GST-fusion protein (panel F ) passed as analytes over streptavidin–gold nanosensors pre-bound with a negative control biotinylated peptide (rat beta2-adrenergic receptor C-tail) or biotinylated SAPAP3 C-tail peptide AQTRL-COOH. ( G ) Plots of mean ± std. dev. for maximal response (R max in RUs, circles; left y -axis) and for dissociation constant (K D in molar, diamonds; right y -axis); N = 2 for R59Q variant vs. SAPAP3 or MEK2 C-tail biosensors, N = 3 for wildtype PDZ domain vs. MEK2 C-tail biosensor, and N = 4 for wildtype PDZ domain vs. SAPAP3 C-tail biosensor.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Ligand Binding Assay, Variant Assay, Negative Control, Binding Assay, Software, Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Representative results of MD simulations of unliganded and SAPAP3-liganded RGS12 PDZ domain models from AlphaFold2. ( A ) Plots of Cα-backbone root-mean-square deviation (RMSD; Å) over time (nanoseconds) during 600 ns MD simulation trajectories for the protein components of all three AF2-derived protein models (“Apo” = unliganded wildtype RGS12 PDZ domain; “WT” = Holo1 PDZ domain; “MUT” = Holo2 PDZ domain). ( B ) Plots of radius of gyration (rGyr or “Rg”; Å), for the AQTRL-COOH peptide ligand within the wildtype (“WT”) Holo1 complex and the R59Q-variant (“MUT”) Holo2 complex. ( C , D ) Plots of Cα-backbone RMSD over time during the MD trajectory for (panel C ) PDZ domain (“protein”) and AQTRL-COOH ligand components of the wildtype Holo1 model, and for (panel D ) PDZ domain (“protein”) and AQTRL-COOH ligand components of the R59Q-variant Holo2 model. ( E ) Solvent-accessible surface area (SASA; Å 2 ) of the wildtype RGS12 PDZ domain (“WT” = Holo1 complex) or R59Q-variant PDZ domain (“MUT” = Holo2 complex) during the MD trajectories. ( F ) Root-mean-square fluctuation plots (RMSF; Å) for each residue of the wildtype ("WT") and R59Q-variant (“MUT”) RGS12 PDZ domain bound to AQTRL-COOH during the 600 ns MD simulation. RMSF values reflect the flexibility of each amino acid position over the course of the simulation, with larger RMSF values indicating higher atomic fluctuation. Both the wildtype and mutant PDZ domains exhibited regions of heightened flexibility, particularly at the N-terminal and C-terminal residues, although overall trends in flexibility were similar between the two models. Per-residue fluctuation was on average only 1.25% higher in Holo2.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Derivative Assay, Variant Assay, Solvent, Residue, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

doi: 10.3390/ijms252111431

Figure Lengend Snippet: Predicted differences in hydrogen bond occupancy of the G-Φ-G-Φ motif during MD trajectory for elements of the final amino acid (“Leu-0”) and carboxylate moiety of the SAPAP3 ligand (AQTRL-COOH). Values obtained for the wildtype RGS12 PDZ domain model (Holo1) are in black brackets; values for the R59Q variant model (Holo2) are in magenta.

Article Snippet: Wildtype and R59Q-variant versions of the human RGS12 PDZ domain (aa 18–101 of UniProt O14924) were each cloned in frame with an N-terminal glutathione-S-transferase (GST) expression tag, expressed by E. coli fermentation, and purified by GST-affinity column and size-exclusion column chromatographies by GenScript.

Techniques: Variant Assay