Structural Insights into the Interaction between the Bacterial Flagellar Motor Proteins FliF and FliG

The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliGN) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance (NMR) and other techniques, we show that the last ∼40 amino acids of FliF (FliFC) interact strongly (upper bound Kd in the low nanomolar range) with FliGN. The formation of this complex causes extensive conformational changes in FliGN. We find that T. maritima FliGN is homodimeric in the absence of the FliFC peptide but forms a heterodimeric complex with the peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG, as well. We relate previously observed phenotypic effects of FliFC mutations to our direct observation of binding. Lastly, on the basis of NMR data, we propose that the primary interaction site for FliFC is located on a conserved hydrophobic patch centered along helix 1 of FliGN. These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring’s precise molecular structure and mechanism of rotational switching

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

Neuronal calcium sensor-1 (NCS-1) is a small calcium binding protein that plays a key role in the internalization and desensitization of activated D2 dopamine receptors (D2Rs). Here, we have used fluorescence anisotropy (FA) and a panel of NCS-1 EF-hand variants to interrogate the interaction between the D2R and NCS-1. Our data are consistent with the following conclusions. (1) FA titration experiments indicate that at low D2R peptide concentrations calcium-loaded NCS-1 binds to the D2R peptide in a monomeric form. At high D2R peptide concentrations, the FA titration data are best fit by a model in which the D2R peptide binds two NCS-1 monomers sequentially in a cooperative fashion. (2) Competition FA experiments in which unlabeled D2R peptide was used to compete with labeled peptide for binding to NCS-1 shifted titration curves to higher NCS-1 concentrations, suggesting that the binding of NCS-1 to the D2R is highly specific and that binding occurs in a cooperative fashion. (3) N-Terminally myristoylated NCS-1 dimerizes in a calcium-dependent manner. (4) Co-immunoprecipitation experiments in HEK-293 confirm that NCS-1 can oligomerize in cell lysates and that oligomerization is dependent on calcium binding and requires functionally intact EF-hand domains. (5) Ca2+/Mg2+ FA titration experiments revealed that NCS-1 EF-hands 2–4 (EF2–4) contributed to binding with the D2R peptide. EF2 appears to have the highest affinity for Ca2+, and occupancy of this site is sufficient to promote high-affinity binding of the NCS-1 monomer to the D2R peptide. Magnesium ions may serve as a physiological cofactor with calcium for NCS-1–D2R binding. Finally, we propose a structural model that predicts that the D2R peptide binds to the first 60 residues of NCS-1. Together, our results support the possibility of using FA to screen for small molecule drugs that can specifically block the interaction between the D2R and NCS-1

Identification of novel MORIPs.

1<p>MORIPs were identified using either the full-length mu-opioid receptor (MOR) or second intracellular loop of the MOR (IL2) as baits in MYTH or traditional Y2H screens, respectively.</p>2<p>Residues indicate the amino acid residues of the protein fragments recovered in the Y2H screens.</p

Confirmation of MOR-MORIP interactions.

1<p>MORIPs isolated either in traditional or MYTH screens were tested for interaction with each of the intracellular loops (IL1, 2, or 3) or carboxyl-terminus (C-tail) of the MOR in a directed Y2H assay.</p>2<p>GST PD: Full-length MORIPs were tested for interaction with the MOR-IL2 in a GST pulldown assay.</p>3<p>Co-IP: Full-length MORIPs were tested for association with the delta (DOR), kappa (KOR), or mu (MOR) opioid receptor by co-immunoprecipitation.</p>+<p>indicates a positive result, - indicates a negative result, while ND indicates not done.</p

Confirmation of MORIP/MOR-IL2 interactions.

<p>GST pulldown assays were performed to interrogate the interaction between selected full-length MORIPs and the MOR-IL2 domain. In the top three panels, MORIP-GST fusion proteins were used to pull down the S-tagged MOR-IL2. In the bottom three panels, MOR-IL2-GST fusion proteins were used to pull down S-tagged MORIPs. Pulldown products were purified on glutathione beads, separated by SDS-PAGE, and probed on Western blots using HRP-conjugated anti-S-tag antibodies. S-tagged MORIPs or MOR-IL2 domains produced in bacteria are shown in lysate lanes (Ly), while uncoated glutathione sepharose beads (Beads) or GST-coated glutathione sepharose beads (GST) incubated with S-tagged proteins served as negative controls. PD indicates pull-down lanes.</p

Deletion of C443 affects stability of the D2R.

<p>(A, B) FLAG-tagged WT-D2R or ΔC443-D2R cDNAs were transiently expressed in HEK-293T cells. Quantitation of receptor expression was normalized to GAPDH expression for protein and mRNA. (A) Proteins were separated by SDS-PAGE and analyzed by Western blotting (left). WT-D2R and ΔC443-D2R mRNA expression levels were determined by RT-qPCR and normalized to wild-type levels (bottom graph). The top bar graph represents the average pixel density (as determined by ImageJ) from three separate experiments. All data were analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 3, **P<0.01). (B) Cells were treated with 50 μg/mL cycloheximide for the indicated times. Proteins were separated by SDS-PAGE and analyzed by Western blotting (top). The bar graph represents the average pixel density (as determined by ImageJ) from four separate experiments. Data were analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 4, *P < 0.05, **P<0.01).</p

C443 is the major site of palmitoylation of the D2R.

<p>(A) Schematic representation of D2L showing the location of cysteine residue mutations. (B) FLAG-tagged WT-D2R or D2R mutants were transiently expressed in HEK-293T cells and expression in cell lysates was analyzed by Western blotting. Untransfected cells served as controls. (C) Normalized amounts of 15-HDYA-labeled D2Rs were IP’d and fluorescently imaged at 725 PMT (top) and 760 PMT (bottom). (D) Total D2R from IPs was analyzed by Western blotting. (E) Levels of palmitoylated D2Rs were normalized to the amount of IP’d receptor. Brackets indicate bands corresponding to D2R that were used for quantitation. The bar graph represents the average pixel density (as determined by ImageJ) from five separate experiments. Data were analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 5, ***P < 0.001).</p

MORIP expression in brain regions of morphine-treated mice.

<p>Mice were treated for 96 hours with either morphine-containing (n = 5) or placebo (n = 4) pellets. Animals were sacrificed, brain regions dissected, and Western blots of selected MORIPs probed with MORIP-specific antibodies. Each panel contains a representative blot for a MORIP in the specified brain region (n = 4 blots/MORIP/brain region). Total protein was quantified by Ponceau stain of the blot prior to antibody probing. Bar graphs represent the average pixel density (as determined by imageJ) of four blots for each brain region normalized to total protein and placebo treatment. Data was analyzed using a two-sided Student’s t-test. Error is expressed as standard error of the mean. * indicates a statistically significant difference (p<.05) between sham and morphine treatment.</p

Role of SIAH1 and SIAH2 in regulating MOR ubiquitination.

<p>(A) Mapping the interaction site of MOR-IL2 on SIAH1 using GST-pulldowns. As controls, S-tagged constructs were incubated with untreated or GST-coated beads. (B) HEK-MOR cells were transfected with wild-type or truncated SIAH (trSIAH1 or trSIAH2) constructs and treated for 6 hours with 30 µM MG132. Proteins were immunoprecipitated and blots probed with either mouse anti-myc or mouse anti-HA to test for SIAH expression (left panels) and interaction with MOR (right panels), respectively. (C) HEK-MOR cells were transfected with wild-type or truncated SIAH constructs and either left untreated or treated with 10 µM DADLE for 6 hours. Blots were cut into sections and probed with rabbit anti-FLAG, mouse anti-myc, mouse anti-HA, or chicken anti-GAPDH antibodies. Bar graphs represent the average pixel density from 4 experiments normalized to GAPDH and untreated controls and subjected to a two-sided paired Student’s t-test. None of the SIAH constructs caused significant changes (at p<.05) in steady-state levels of MOR protein expression or in DADLE-mediated decreases in MOR expression levels. (D–F) Ubiquitination of MOR. Equal amounts of MOR (normalized from lysate blot) were loaded into immunoprecipitation reactions with anti-FLAG antibody. In each panel, the upper blot shows the IP probed for ubiquitin. All other blots show expression of various constructs or GAPDH in transfected cells. All experiments were performed in triplicate. (D) Steady-state ubiquitination levels in transfected HEK-MOR cells (E) Ubiquitination levels in transfected cells treated with 30 µM MG132 (F) Agonist induced ubiquitination in transfected cells treated with 10 µM DADLE.</p