Na ؉ -H ؉ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodiumphosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1␣ (PP1␣), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 . Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1␣-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ␤-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally . 4 The abbreviations used are: PTH, parathyroid hormone; PTHR, PTH receptor; NHERF1, Na ϩ /H ϩ -exchanger regulatory factor 1; GnTI Ϫ , N-acetylglucosaminyltransferase-deficient HEK-293S cells; TAP-NHERF1, tandem affinity purification-tagged NHERF1; FLIM, fluorescence lifetime imaging; HDX, hydrogen/deuterium exchange mass spectrometry; pSer 290 , phosphorylated Ser 290 ; NP40, Nonidet P-40; TAMRA, carboxytetramethylrhodamine; SILAC, stable isotope labeling of amino acids in cell culture; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ND, nondeuterated; FD, fully deuterated; EBD, ezrin-binding domain; PDB, Protein Data Bank; 2Me-4OMe-TM, 7-hydroxy-5,5-dimethyl-10-(4-methoxy-2-methylphenyl)-dibenzo-[b,e]-silin-3(5H)-one; ANOVA, analysis of variance; ID, intrinsically disordered; TTN, tautomycetin; RPTEC, renal proximal tubule epithelial cell; pen/strep, penicillin and streptomycin; CFP, cyan fluorescent protein; FERM, Ezrin-Radixin-Moesin; MD, molecular dynamics.

Proteins are held together in the native state by hydrophobic interactions, hydrogen bonds and interactions with the surrounding water, whose strength as well as spatial and temporal distribution affects protein flexibility and hence function. We study these effects using 10 ns molecular dynamics simulations of pure water and of two proteins, the glutamate receptor ligand binding domain and barnase. We find that most of the noncovalent interactions flicker on and off over typically nanoseconds, and so we can obtain good statistics from the molecular dynamics simulations. Based on this information, a topological network of rigid bonds corresponding to a protein structure with covalent and noncovalent bonds is constructed, with account being taken of the influence of the flickering hydrogen bonds. We define the duty cycle for the noncovalent interactions as the percentage of time a given interaction is present, which we use as an input to investigate flexibility/rigidity patterns, in the algorithm FIRST which constructs and analyses topological networks.

Background: Some inherited defects in NHERF1 are associated with high phosphate (P i ) excretion and skeletal abnormalities. Results: NHERF1 forms a multiprotein complex with Npt2a and ezrin. Mutants fail to assemble this ternary complex. Conclusion:The NHERF1 ternary complex is required for PTH-sensitive P i transport. Significance: NHERF1 mutations may cause disease processes through structural changes that prevent assembly of multiprotein complexes.

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar K Ds. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.

The Na+/H+ exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The parathyroid hormone receptor (PTHR), type II sodium-dependent phosphate co-transporter (Npt2a), and β2-adrenergic receptor (β2-AR), through their respective carboxy-terminal PDZ recognition motifs, individually interact with NHERF1 forming a complex with one of the PDZ domains. In the basal state, NHERF1 adopts a self-inhibited conformation, in which its carboxy-terminal PDZ ligand interacts with PDZ2. We applied Molecular Dynamics (MD) simulations to uncover the structural and biochemical basis for the binding selectivity of NHERF1 PDZ domains. PDZ1 uniquely forms several contacts not present in PDZ2 that further stabilize PDZ1 interactions with target ligands. The binding free energy (ΔG) of PDZ1 and PDZ2 with the carboxy-terminal, five-amino acid residues that form the PDZ-recognition motif of PTHR, Npt2a, and β2-AR was calculated and compared with the calculated ΔG for the self-association of NHERF1. The results suggest that the interaction of the PTHR, β2-adrenergic, and Npt2a involves competition between NHERF1 PDZ domains and the target proteins. The binding of PDZ2 with PTHR may also compete with the self-inhibited conformation of NHERF1, thereby contributing to the stabilization of an active NHERF1 conformation.