The binding affinities of proteins interacting with the PDZ domain of PICK1

Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo-1 (PDZ) protein-protein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target.     

KIN‐4/MAST kinase promotes PTEN‐mediated longevity of Caenorhabditis elegans via binding through a PDZ domain

PDZ domain‐containing proteins (PDZ proteins) act as scaffolds for protein–protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN‐4, a PDZ domain‐containing microtubule‐associated serine‐threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin‐4 is required for the long lifespan of daf‐2/insulin/IGF‐1 receptor mutants. We then show that neurons are crucial tissues for the longevity‐promoting role of kin‐4. We find that the PDZ domain of KIN‐4 binds PTEN, a key factor for the longevity of daf‐2 mutants. Moreover, the interaction between KIN‐4 and PTEN is essential for the extended lifespan of daf‐2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age‐related diseases in mammals through their interaction with PTEN.     

Regulation of hemicholinium binding sites in isolated nerve terminals

High-affinity uptake of choline, the rate-limiting, regulatory step for the synthesis of acetylcholine is regulated via presynaptic auto- and heteroreceptors. Binding studies using tritiated hemicholinium-3 ([³H]HCh-3) as the specific ligand for the choline carrier revealed that the number of hemicholinium binding sites in nerve terminals isolated from insect brain changes corresponding to the activity of synaptosomal kinase A and kinase C. Activation of kinase A apparently increases the total number of hemicholinium binding sites by recruiting additional occult carriers, whereas the effect of kinase C activity is most appropriately explained by preventing a down-regulation of carrier proteins. The kinase-mediated regulation of choline transporters is obviously due to a phosphorylation of the carrier protein itself.     

The structural flexibility of the shank1 PDZ domain is important for its binding to different ligands

The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3 Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain.     

NEMO binding domain of IKK-2 encompasses amino acids 735-745

NF-kappaB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK-1, IKK-2, and NEMO/IKKgamma, it is the interaction between IKK-2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C-terminal LDWSWL residues of IKK-2 (called the Nemo Binding Domain (NBD)) and the N-terminus of NEMO. In an effort to characterize the IKK-2:NEMO interaction, we have investigated several NBD-containing peptides for their ability to bind NEMO and inhibit the critical IKK-2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK-2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD-containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD-containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO.     

Brain-derived neurotrophic factor signal enhances and maintains the expression of AMPA receptor-associated PDZ proteins in developing cortical neurons

Postsynaptic molecules with PDZ domains (PDZ proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. In rat neocortical development, the protein expression of AMPA-type glutamate receptor GluR1 lagged behind its mRNA expression and rather paralleled an increase in PDZ protein levels. One of the neurotrophins, brain-derived neurotrophic factor (BDNF), appeared to contribute to this process, regulating the PDZ protein expression. In neocortical cultures, BDNF treatment upregulated SAP97, GRIP1, and Pick1 PDZ proteins. Conversely, BDNF gene targeting downregulated these same PDZ molecules. The BDNF-triggered increases in PDZ proteins resulted in the elevation of their total association with the AMPA receptors GluR1 and GluR2/3, which led to the increase in AMPA receptor proteins. When Sindbis viruses carrying GluR1 or GluR2 C-terminal decoys disrupted their interactions, GluR2 C-terminal decoys inhibited both BDNF-triggered GluR1 and GluR2/3 increases, whereas GluR1 C-terminal decoys blocked only the BDNF-triggered GluR1 increase. In agreement, coexpression of SAP97 and GluR1 in nonneuronal HEK293 cells increased both proteins compared with their single transfection, implying mutual stabilization. This work reveals a novel function of BDNF in postsynaptic development by regulating the PDZ protein expression.     

Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies

Human Chk2 is a newly identified tumor suppressor protein involved in signaling pathways in response to DNA damage. The protein consists of a forkhead-associated (FHA) domain and a kinase domain. Identification of binding partners of the Chk2FHA domain is important in understanding the roles of Chk2 in signaling. We report development of an approach involving the use of combinatorial libraries, pull-down assays, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) methods to identify possible candidates for the binding sites of Chk2FHA. The approach has been used to identify Thr329 of p53 and Thr1852 of breast cancer type 1 susceptibility protein (BRCA1) as very likely biological binding sites of Chk2FHA. The results provide useful leads for further biological analyses of cell signaling involving the FHA domain of Chk2 protein.     

Identification of functional nuclear export sequences in human sphingosine kinase 1

Sphingosine kinase (SPHK) is an enzyme that phosphorylates sphingosine to form sphingosine 1-phosphate (S1P). Human SPHK1 (hSPHK1) was localized predominantly in the cytoplasm when transiently expressed in Cos7 cells. In this study, we have found two functional nuclear export signal (NES) sequences in the middle region of hSPHK1. Deletion and mutagenesis studies revealed that the cytoplasmic localization of SPHK1 depends on its nuclear export, directed by the NES. Furthermore, upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of hSPHK1 was observed, indicating that hSPHK1 shuttles between the cytoplasm and the nucleus. Our results provide the first evidence of the active nuclear export of SPHK1 and suggest it is mediated by a CRM1-dependent pathway.     

Role of binding proteins to IRS-1 in insulin signalling

Insulin elicits its divergent metabolic and mitogenic effects by binding to its specific receptor, which belongs to the family of receptor tyrosine kinases. The activated insulin receptor phosphorylates the intracellular substrate IRS-1, which then binds various signalling molecules that contain SRC homology 2 domains, thereby propagating the insulin signal. Among these IRS-1-binding proteins, the Grb2-Sos complex and the protein tyrosine phosphatase SHP-2 transmit mitogenic signals through the activation of Ras, and phosphoinositide 3-kinase is implicated in the major metabolic actions of insulin. Although substantial evidence indicates the importance of IRS-1 in insulin signal transduction, the generation of IRS-1-deficient mice has revealed the existence of redundant signalling pathways.     

Identification of the critical structural determinants of the EF-hand domain arrangements in calcium binding proteins

EF-hand calcium binding proteins (CaBPs) share strong sequence homology, but exhibit great diversity in structure and function. Thus although calmodulin (CaM) and calcineurin B (CNB) both consist of four EF hands, their domain arrangements are quite distinct. CaM and the CaM-like proteins are characterized by an extended architecture, whereas CNB and the CNB-like proteins have a more compact form. In this study, we performed structural alignments and molecular dynamics (MD) simulations on 3 CaM-like proteins and 6 CNB-like proteins, and quantified their distinct structural and dynamical features in an effort to establish how their sequences specify their structures and dynamics. Alignments of the EF2-EF3 region of these proteins revealed that several residues (not restricted to the linker between the EF2 and EF3 motifs) differed between the two groups of proteins. A customized inverse folding approach followed by structural assessments and MD simulations established the critical role of these residues in determining the structure of the proteins. Identification of the critical determinants of the two different EF-hand domain arrangements and the distinct dynamical features relevant to their respective functions provides insight into the relationships between sequence, structure, dynamics and function among these EF-hand CaBPs.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [³H]-leukotriene D₄ ([³H]-LTD₄), in the presence or absence of unlabeled LTD₄, the diastereoisomer of LTD₄ (5R,6S-LTD₄), leukotriene E₄ (LTE₄) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [³H]-LTD₄ in the crude membrane fraction isolated from guinea pig lung. The time required for [³H]-LTD₄ binding to reach equilibrium was approximately 20 to 25 min at 37°C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [³H]-LTD₄ to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (B_max), derived from Scatchard analysis, was approximately 320±200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5±4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD₄, FPL 55712 and 5R,6S-LTD₄ competed with [³H]-LTD₄. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD₄, did not significantly compete with [³H]-LTD₄ at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

Stereoselective binding of zopiclone to human plasma proteins

The binding of racemic zopiclone (ZOP) and of its two enantiomers to plasma proteins, albumin and α1‐acid glycoprotein were compared. Our work shows that the binding of ZOP to human plasma proteins is stereoselective. The total plasma protein binding percentages were 79.3 ± 5.5%, 83.8 ± 5.2%, and 75.1 ± 2.1%, for racemic zopiclone, (−)zopiclone and (+)zopiclone, respectively. These results were confirmed by the analysis of samples obtained from healthy volunteers after the oral administration of ZOP. The anticoagulant used for sampling was also shown to have an influence on the percentage binding and on its stereoselectivity. Considering albumin and α1‐acid glycoprotein separately, stereoselectivity was also observed. Chirality 11:129–132, 1999. © 1999 Wiley‐Liss, Inc.     

Structural and functional differences of SWIRM domain subtypes

SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.     

Identification of hyaluronic acid binding sites in the extracellular domain of CD44

CD44 is a polymorphic glycoprotein expressed on the surface of many tissues and cell lines which has been implicated in a number of cellular functions including lymphocyte homing to mucosal lymphoid tissue (Peyers patches), leukocyte activation, lymphopoiesis, and tumor metastasis. The predominant isoform found on human leukocytes, CD44H, is a receptor for hyaluronic acid. Because of the prominent role CD44 plays in diverse biological processes, we set out to identify the hyaluronic acid binding site(s) in the extracellular domain of CD44H. Using truncation and site-directed mutagenesis we identified two regions containing clusters of conserved basic residues which are important in hyaluronic acid binding. One of these regions is situated near the NH2 terminus and is homologous to other hyaluronic acid binding proteins including cartilage link protein. The other more membrane proximal region lies outside the link protein homologous domain. Mutagenesis of basic residues within these regions established their role as determinants in hyaluronic acid binding. Mutation of Arg 41, a position where a basic residue is conserved in all hyaluronic acid binding proteins, completely abolished binding suggesting that this residue plays a critical role in hyaluronic acid binding.     

Identification of nucleoside transport binding sites in the human myocardium

The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals.Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation.Equilibrium binding assays were performed using [³H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (B_max) and dissociation constant (K_d) of [³H]-NBMPR binding of human myocardial preparations.Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a B_max of 283 ± 32 fmol/mg protein and K_d of 0.56 ± 0.12 nM. These values are lower than those obtained from guinea pigs (B_max = 1440 ± 187 fmol/mg protein and K_d = 0.21 ± 0.03 nM) and canine atrium (B_max 594 ± 73 fmol/mg protein, and K_d = 1.12 ± 0.22 nM).Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes.     

Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand

Soluble Fas ligand (sFasL) is released from the cell surface by matrix metalloproteinases (MMPs), one of which is MMP-7. We have reported that MMP-7-generated sFasL is pro-apoptotic in both in vitro and in vivo systems. However, there are contradictory reports that the soluble form of FasL is inactive or anti-apoptotic, resulting in significant controversy in the literature. One potential explanation for these discrepancies is that forms of sFasL with different amino-terminal sequences have been demonstrated to have varying activities. Here we report that MMP-7 cleaves murine and human FasL at sites that are distinct from previously reported cleavage sites resulting in production of novel forms of sFasL. Cleavage of FasL by MMP-7 occurs at the leucine residues in the sequence "ELAELR" within the region between the transmembrane and trimerization domains. When this site is unavailable, a more c-terminal site, "SL," is cleaved. MMP-7 differentially processes murine and human FasL since it cleaves human FasL not only at the "ELAELR" site but also at a previously identified site. Additionally, MMP-3, but not MMP-2, was found to have the same cleavage specificity for murine FasL as MMP-7. We conclude that the controversy regarding the biological activity of sFasL may be explained, in part, by the generation of distinct forms of sFasL as a result of cleavage at specific sequences.