Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Background & Aims: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation. Methods: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set. Results: SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy. Conclusions: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.

Targeting epithelial to mesenchymal transition in prostate cancer by a novel compound, plectranthoic acid, isolated from Ficus microcarpa.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in prostate cancer (PCa) metastasis. This has led to a surge in the efforts for identification of safer and more effective compounds which can modulate EMT and consequently inhibiting migration and invasion of PCa cells. We reported previously that Plectranthoic acid (PA), a natural compound isolated from the extracts of Ficus microcarpa, has the capability to induce cell cycle arrest and apoptosis in PCa cells. Here, we determined the effects of PA on EMT, migration, and invasion of PCa cells. Inhibition of EMT induced by different mitogens was effectively inhibited by PA treatment with subsequent decrease in migration of PCa cells. Employing a PCa cell culture model of TGF-ß-induced EMT, we showed that PA has the ability to reverse EMT. PA treatment was associated with induction of epithelial markers and decrease in the expression of mesenchymal markers in PCa cells. Proteomic analysis identified Rac1 as the major cadherin signaling protein modulated with PA treatment. In silico studies indicated that PA docked to the CH domain of NEDD9 protein with an estimated free binding energy of -7.34 Kcal/moL. Our studies revealed significant inhibition of Rac1/NEDD9 pathway in PA treated cells thereby providing a molecular basis of the inhibitory effect of PA on PCa cell migration and invasion. In conclusion, our data suggest that PA should be investigated further as an adjuvant treatment in human PCa cells, given its potential as an anti-invasive agent.

Quinoline-based Protein-protein Interaction Inhibitors of LEDGF/p75 and HIV Integrase: An In Silico Study.

The failure of the Integrase Strand Transfer Inhibitors (INSTIs) due to the mutations occurring at the catalytic site of HIV integrase (IN) has led to the design of allosteric integrase inhibitors (ALLINIs). Lens epithelium derived growth factor (LEDGF/p75) is the host cellular cofactor which helps chaining IN to the chromatin. The protein-protein interactions (PPIs) were observed at the allosteric site (LEDGF/p75 binding domain) between LEDGF/p75 of the host cell and IN of virus. In recent years, many small molecules such as CX04328, CHIBA-3053 and CHI-104 have been reported as LEDGF/p75-IN interaction inhibitors (LEDGINs). LEDGINs have emerged as promising therapeutics to halt the PPIs by binding at the interface of both the proteins. In the present work, we correlated the docking scores for the reported LEDGINs containing quinoline scaffold with the in vitro biological data. The hierarchal clustering method was used to divide the compounds into test and training set. The robustness of the generated model was validated by q2 and r2 for the predicted set of compounds. The generated model between the docking score and biological data was assessed to predict the activity of the hits (quinoline scaffold) obtained from virtual screening of LEDGINs providing their structureactivity relationships to aim for the generation of potent agents.

DYRK1A regulates Hap1-Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome.

Huntingtin-associated protein 1 (Hap1) is known to be critical for postnatal hypothalamic function and growth. Hap1 forms stigmoid bodies (SBs), unique neuronal cytoplasmic inclusions of unknown function that are enriched in hypothalamic neurons. Here we developed a simple strategy to isolate the SB-enriched fraction from mouse brain. By analyzing Hap1 immunoprecipitants from this fraction, we identified a Hap1-interacting SB component, DDB1 and CUL4 associated factor 7 (Dcaf7)/WD40 repeat 68 (WDR68), whose protein level and nuclear translocation are regulated by Hap1. Moreover, we found that Hap1 bound Dcaf7 competitively in cytoplasm with dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), a protein implicated in Down syndrome (DS). Depleting Hap1 promoted the DYRK1A-Dcaf7 interaction and increased the DYRK1A protein level. Transgenic DS mice overexpressing DYRK1A showed reduced Hap1-Dcaf7 association in the hypothalamus. Furthermore, the overexpression of DYRK1A in the hypothalamus led to delayed growth in postnatal mice, suggesting that DYRK1A regulates the Hap1-Dcaf7 interaction and postnatal growth and that targeting Hap1 or Dcaf7 could ameliorate growth retardation in DS.

Evaluating p97 Inhibitor Analogues for Potency against p97-p37 and p97-Npl4-Ufd1 Complexes.

We previously found that the p97 cofactor, p47, significantly decreased the potency of some ATP-competitive p97 inhibitors such as ML240 [2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine] and ML241 [2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8 tetrahydroquinazolin-4-amine]. In this study, we aimed to evaluate inhibitor potencies against two additional p97 cofactor complexes, p97-p37 and p97-Npl4-Ufd1. We focused on these two cofactor complexes, because the protein sequence of p37 is 50 % identical to that of p47, and the Npl4-Ufd1 heterodimer (NU) is the most-studied p97 cofactor complex. We screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of p97 alone and of p97-p37 and p97-NU complexes. In contrast to the effect of p47, p37 and NU did not significantly change the potencies of most of the compounds. These results highlight differences among p97 cofactors in influencing p97 conformation and effects of inhibitors on p97 complexes, as compared to p97 alone. Continued efforts are needed to advance the development of complex-specific p97 inhibitors.

Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.

In this study, capillary electrophoresis (CE) combined with HPLC-MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC-MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentation information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC-MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors.

The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment.

The Hippo pathway is an important organ size control signaling network and the major regulatory mechanism of cell-contact inhibition. Yes associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are its targets and terminal effectors: inhibition of the pathway promotes YAP/TAZ translocation to the nucleus, where they interact with transcriptional enhancer associate domain (TEAD) transcription factors and coactivate the expression of target genes, promoting cell proliferation. Defects in the pathway can result in overgrowth phenotypes due to deregulation of stem-cell proliferation and apoptosis; members of the pathway are directly involved in cancer development. The pharmacological regulation of the pathway might be useful in cancer prevention, treatment, and regenerative medicine applications; currently, a few compounds can selectively modulate the pathway. In this review, we present an overview of the Hippo pathway, the sequence and structural analysis of YAP/TAZ, the known pharmacological modulators of the pathway, especially those targeting YAP/TAZ-TEAD interaction.

Changes in vitamin D target gene expression in adipose tissue monitor the vitamin D response of human individuals.

SCOPE: Vitamin D3, its biologically most active metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), and the vitamin D receptor (VDR) are important for adipose tissue biology. METHODS AND RESULTS: We extrapolated genomic VDR association loci in adipocytes from 55 conserved genome-wide VDR-binding sites in nonfat tissues. Taking the genes DUSP10, TRAK1, NRIP1, and THBD as examples, we confirmed the predicted VDR binding sites upstream of their transcription start sites and showed rapid mRNA up-regulation of all four genes in SGBS human pre-adipocytes. Using adipose tissue biopsy samples from 47 participants of a 5-month vitamin D3 intervention study, we demonstrated that all four primary VDR target genes can serve as biomarkers for the vitamin D3 responsiveness of human individuals. Changes in DUSP10 gene expression appear to be the most comprehensive marker, while THBD mRNA changes characterized a rather different group of study participants. CONCLUSION: We present a new approach to predict vitamin D target genes based on conserved genomic VDR-binding sites. Using human adipocytes as examples, we show that such ubiquitous VDR target genes can be used as markers for the individual's response to a supplementation with vitamin D3.

Determination of topological structure of ARL6ip1 in cells: identification of the essential binding region of ARL6ip1 for conophylline.

Conophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three-spanning transmembrane protein and determined the CNP-binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl-amino-CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti-apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases.

Phage display-directed discovery of LEDGF/p75 binding cyclic peptide inhibitors of HIV replication.

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.

Complementary proteomic tools for the dissection of apoptotic proteolysis events.

Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein's structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.

Preparation of conophylline affinity nano-beads and identification of a target protein.

Conophylline, a vinca alkaloid extracted from the tropical plant Ervatamia microphylla, has been shown to induce the differentiation of insulin-producing beta-cells in cultured cells and in animals. However, its mechanism of action and the molecular target have remained unclear. Therefore, we prepared a fishing probe with conophylline to identify the target protein by using latex nano-beads, which are newly innovated tools for affinity-purification. With these conophylline-linked nano-beads, we found that conophylline directly interacted with ARL6IP. ARL6IP may thus be involved in the mechanism of cellular differentiation of beta-cells, and this probe should be useful to find other target proteins.

Requirements of SLP76 tyrosines in ITAM and integrin receptor signaling and in platelet function in vivo.

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP76), an adaptor that plays a critical role in platelet activation in vitro, contains three N-terminal tyrosine residues that are essential for its function. We demonstrate that mice containing complementary tyrosine to phenylalanine mutations in Y145 (Y145F) and Y112 and Y128 (Y112/128F) differentially regulate integrin and collagen receptor signaling. We show that mutation of Y145 leads to severe impairment of glycoprotein VI (GPVI)-mediated responses while preserving outside-in integrin signaling. Platelets from Y112/128F mice, although having mild defects in GPVI signaling, exhibit defective actin reorganization after GPVI or alpha IIb beta 3 engagement. The in vivo consequences of these signaling defects correlate with the mild protection from thrombosis seen in Y112/128F mice and the near complete protection observed in Y145F mice. Using genetic complementation, we further demonstrate that all three phosphorylatable tyrosines are required within the same SLP76 molecule to support platelet activation by GPVI.

Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

Crystal structure at 2.8 A of Huntingtin-interacting protein 1 (HIP1) coiled-coil domain reveals a charged surface suitable for HIP1 protein interactor (HIPPI).

Huntington's disease is a genetic neurological disorder that is triggered by the dissociation of the huntingtin protein (htt) from its obligate interaction partner Huntingtin-interacting protein 1 (HIP1). The release of the huntingtin protein permits HIP1 protein interactor (HIPPI) to bind to its recognition site on HIP1 to form a HIPPI/HIP1 complex that recruits procaspase-8 to begin the process of apoptosis. The interaction module between HIPPI and HIP1 was predicted to resemble a death-effector domain. Our 2.8-A crystal structure of the HIP1 371-481 subfragment that includes F432 and K474, which is important for HIPPI binding, is not a death-effector domain but is a partially opened coiled coil. The HIP1 371-481 model reveals a basic surface that we hypothesize to be suitable for binding HIPPI. There is an opened region next to the putative HIPPI site that is highly negatively charged. The acidic residues in this region are highly conserved in HIP1 and a related protein, HIP1R, from different organisms but are not conserved in the yeast homologue of HIP1, sla2p. We have modeled approximately 85% of the coiled-coil domain by joining our new HIP1 371-481 structure to the HIP1 482-586 model (Protein Data Bank code: 2NO2). Finally, the middle of this coiled-coil domain may be intrinsically flexible and suggests a new interaction model where HIPPI binds to a U-shaped HIP1 molecule.

Vitamin B6 conjugation to nuclear corepressor RIP140 and its role in gene regulation.

Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, is an important cofactor in amino acid metabolism, and supplementary vitamin B6 has protective effects in many disorders. Other than serving as a cofactor, it can also modulate the activities of steroid hormone receptors and transcription factors. However, the molecular basis of this modulation is unclear. Here, we report that mouse nuclear receptor interacting protein 140 (RIP140) can be modified by PLP conjugation. We mapped the modification site to Lys613 by LC-ESI-MS/MS analysis. This modification enhanced its transcriptional corepressive activity and its physiological function in adipocyte differentiation. We attribute this effect to increased interaction of RIP140 with histone deacetylases and nuclear retention of RIP140. This study uncovers a new physiological role of vitamin B6 in gene regulation by PLP conjugation to a transcriptional coregulator, which represents a new function of an old form of protein post-translational modification that has important biological consequences.

Rational design of a nonpeptide general chemical scaffold for reversible inhibition of PDZ domain interactions.

Novel small molecules were designed to specifically target the ligand-binding pocket of a PDZ domain. Iterative molecular docking and modeling allowed the design of an indole scaffold 10a as a reversible inhibitor of ligand binding. The 10a scaffold inhibited the interaction between MAGI-3 and PTEN and showed cellular activities that are consistent with the inhibition of NHERF-1 function.

Binding of the intracellular Fas ligand (FasL) domain to the adaptor protein PSTPIP results in a cytoplasmic localization of FasL.

The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL- and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domain-containing adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL.PSTPIP.PTP-PEST complexes which may contribute to FasL reverse signaling.

Solution structure of the second PDZ domain of the neuronal adaptor X11alpha and its interaction with the C-terminal peptide of the human copper chaperone for superoxide dismutase.

Protection against reactive oxygen species is provided by the copper containing enzyme superoxide dismutase 1 (SOD1). The copper chaperone CCS is responsible for copper insertion into apo-SOD1. This role is impaired by an interaction between the second PDZ domain (PDZ2alpha) of the neuronal adaptor protein X11alpha and the third domain of CCS (McLoughlin et al. (2001) J. Biol. Chem., 276, 9303-9307). The solution structure of the PDZ2alpha domain has been determined and the interaction with peptides derived from CCS has been explored. PDZ2alpha binds to the last four amino acids of the CCS protein (PAHL) with a dissociation constant of 91 +/- 2 microM. Peptide variants have been used to map the interaction areas on PDZ2alpha for each amino acid, showing an important role for the C-terminal leucine, in line with canonical PDZ-peptide interactions.