Biophysical and structural analyses of the interaction between the SHANK1 PDZ domain and an internal SLiM.

The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterized, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) - EESTSFQGP - as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM - GKAP - FITC-Ahx-EAQTRL-COOH - the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy. To gain further insight into the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganize to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.

Understanding the molecular basis of folding cooperativity through a comparative analysis of a multidomain protein and its isolated domains.

Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multidomain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multidomain protein may either fold independently on each other or exhibit interdependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single cooperative unit. By exploiting quantitatively, the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short autoinhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multidomain systems.

Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3.

The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to the multi-PDZ domain containing protein Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Here, we used a biochemical reconstitution approach with purified, intact Par complex and qualitative binding assays and found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. The energetic coupling arises from interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) that mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity.

RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein.

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A-NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A-NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.

Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.

The animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3-Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3-Par complex assembly.

Real-time observation of ligand-induced allosteric transitions in a PDZ domain.

While allostery is of paramount importance for protein regulation, the underlying dynamical process of ligand (un)binding at one site, resulting time evolution of the protein structure, and change of the binding affinity at a remote site are not well understood. Here the ligand-induced conformational transition in a widely studied model system of allostery, the PDZ2 domain, is investigated by transient infrared spectroscopy accompanied by molecular dynamics simulations. To this end, an azobenzene-derived photoswitch is linked to a peptide ligand in a way that its binding affinity to the PDZ2 domain changes upon switching, thus initiating an allosteric transition in the PDZ2 domain protein. The subsequent response of the protein, covering four decades of time, ranging from ∼1 ns to ∼µs, can be rationalized by a remodeling of its rugged free-energy landscape, with very subtle shifts in the populations of a small number of structurally well-defined states. It is proposed that structurally and dynamically driven allostery, often discussed as limiting scenarios of allosteric communication, actually go hand-in-hand, allowing the protein to adapt its free-energy landscape to incoming signals.

Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases.

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein-protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.

Domain Analysis and Motif Matcher (DAMM): A Program to Predict Selectivity Determinants in Monosiga brevicollis PDZ Domains Using Human PDZ Data.

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.

Heparanase Involvement in Exosome Formation.

Exosomes are secreted vesicles involved in signaling processes. The biogenesis of a class of these extracellular vesicles depends on syntenin, and on the interaction of this cytosolic protein with syndecans. Heparanase, largely an endosomal enzyme, acts as a regulator of the syndecan-syntenin-exosome biogenesis pathway. The upregulation of syntenin and heparanase in cancers may support the suspected roles of exosomes in tumor biology.

Carboxy-terminal deletion of the HDL receptor reduces receptor levels in liver and steroidogenic tissues, induces hypercholesterolemia, and causes fatal heart disease.

The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis.

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand-binding site of the second PDZ domain of PSD-95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the ß1-ß2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.

Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries.

RATIONALE: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. OBJECTIVE: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation. METHODS AND RESULTS: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. CONCLUSIONS: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.

Deciphering the unconventional peptide binding to the PDZ domain of MAST2.

Phosphatase and tensin homologue (PTEN) and microtubule-associated serine threonine kinase 2 (MAST2) are key negative regulators of survival pathways in neuronal cells. The two proteins interact via the PDZ (PSD-95, Dlg1, Zo-1) domain of MAST2 (MAST2-PDZ). During infection by rabies virus, the viral glycoprotein competes with PTEN for interaction with MAST2-PDZ and promotes neuronal survival. The C-terminal PDZ-binding motifs (PBMs) of the two proteins bind similarly to MAST2-PDZ through an unconventional network of connectivity involving two anchor points. Combining stopped-flow fluorescence, analytical ultracentrifugation (AUC), microcalorimetry and NMR, we document the kinetics of interaction between endogenous and viral ligands to MAST2-PDZ as well as the dynamic and structural effects of these interactions. Viral and PTEN peptide interactions to MAST2-PDZ occur via a unique kinetic step which involves both canonical C-terminal PBM binding and N-terminal anchoring. Indirect effects induced by the PBM binding include modifications to the structure and dynamics of the PDZ dimerization surface which prevent MAST2-PDZ auto-association. Such an energetic communication between binding sites and distal surfaces in PDZ domains provides interesting clues for protein regulation overall.

Protein interacting with C-kinase 1 (PICK1) binding promiscuity relies on unconventional PSD-95/discs-large/ZO-1 homology (PDZ) binding modes for nonclass II PDZ ligands.

PDZ domain proteins control multiple cellular functions by governing assembly of protein complexes. It remains unknown why individual PDZ domains can bind the extreme C terminus of very diverse binding partners and maintain selectivity. By employing NMR spectroscopy, together with molecular modeling, mutational analysis, and fluorescent polarization binding experiments, we identify here three structural mechanisms explaining why the PDZ domain of PICK1 selectively binds >30 receptors, transporters, and kinases. Class II ligands, including the dopamine transporter, adopt a canonical binding mode with promiscuity obtained via differential packing in the binding groove. Class I ligands, such as protein kinase Cα, depend on residues upstream from the canonical binding sequence that are likely to interact with flexible loop residues of the PDZ domain. Finally, we obtain evidence that the unconventional ligand ASIC1a has a dual binding mode involving a canonical insertion and a noncanonical internal insertion with the two C-terminal residues forming interactions outside the groove. Together with an evolutionary analysis, the data show how unconventional binding modes might evolve for a protein recognition domain to expand the repertoire of functionally important interactions.

Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove.

Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

PDZ Domain in the Engineering and Production of a Saporin Chimeric Toxin as a Tool for targeting Cancer Cells.

In this paper we have studied a PDZ protein domain as a possible tool for cellular targeting of the ribosome inactivating protein Saporin, exploiting the ability of PDZ domains to recognize and bind short peptide sequences located at the C-terminus of a cognate protein. We have focused our attention on the PDZ domain from hCASK (Human calcium/calmodulin-dependent serine protein kinase) that binds extracellular CD98 in epithelial cells, being this antigen recognized as a marker for several human tumors and particularly considered a negative prognostic marker for human glioblastoma. We produced recombinant fusions of one or two hCASK-PDZ domains with the ribosome inactivating protein Saporin and assayed them on two human glioblastoma cell lines (GL15 and U87). These constructs proved to be toxic, with increasing activity as a function of the number of PDZ domains, and induce cell death by apoptotic mechanisms in a dose-dependent and/or time dependent manner.

Rigidified clicked dimeric ligands for studying the dynamics of the PDZ1-2 supramodule of PSD-95.

PSD-95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein-protein interactions in the brain with its PDZ domains. It has been suggested that PSD-95 is composed of two supramodules, one of which is the PDZ1-2 tandem domain. Here we have developed rigidified high-affinity dimeric ligands that target the PDZ1-2 supramodule, and established the biophysical parameters of the dynamic PDZ1-2/ligand interactions. By employing ITC, protein NMR, and stopped-flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD-95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.

Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity.

A general strategy was developed for the intracellular delivery of linear peptidyl ligands through fusion to a cell-penetrating peptide and cyclization of the fusion peptides via a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and an inhibitor for the CFTR-associated ligand (CAL) PDZ domain as a potential treatment for cystic fibrosis.

The structures of Arabidopsis Deg5 and Deg8 reveal new insights into HtrA proteases.

Plant Deg5 and Deg8 are two members of the HtrA proteases, a family of oligomeric serine endopeptidases that are involved in a variety of protein quality-control processes. These two HtrA proteases are located in the thylakoid lumen and participate in high-light stress responses by collaborating with other chloroplast proteins. Deg5 and Deg8 degrade photodamaged D1 protein of the photosystem II reaction centre, allowing its in situ replacement. Here, the crystal structures of Arabidopsis thaliana Deg5 (S266A) and Deg8 (S292A) are reported at 2.6 and 2.0 Å resolution, respectively. The Deg5 trimer contains two calcium ions in a central channel, suggesting a link between photodamage control and calcium ions in chloroplasts. Previous structures of HtrA proteases have indicated that their regulation usually requires C-terminal PDZ domain(s). Deg5 is unique in that it contains no PDZ domain and the trimeric structure of Deg5 (S266A) reveals a novel catalytic triad conformation. A similar triad conformation is observed in the hexameric structure of the single PDZ-domain-containing Deg8 (S292A). These findings suggest a novel activation mechanism for plant HtrA proteases and provide structural clues to their function in light-stress response.

Interaction partners of PSD-93 studied by X-ray crystallography and fluorescence polarization spectroscopy.

PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via its PDZ domains. The PDZ domains of PSD-95 and PSD-93 are structurally very similar, but relatively little is known about the function of PSD-93. PSD-93 has been suggested to interact with GluD2 from the family of ionotropic glutamate receptors. Here, the interactions of four residues (GTSI) representing the extreme C-terminus of GluD2 with PSD-93 PDZ1 have been investigated in the crystalline phase. Two different binding modes of these residues were observed, suggesting that the peptide is not tightly bound to PSD-93 PDZ1. In accordance, the two N-terminal PSD-93 PDZ domains show no appreciable binding affinity for a GluD2-derived C-terminal octapeptide, whereas micromolar affinity was observed for a GluN2B-derived C-terminal octapeptide. This indicates that if present, the interactions between GluD2 and PSD-93 involve more than the extreme terminus of the receptor. In contrast, the tumour-suppressor protein SCRIB PDZ3 shows low micromolar affinity towards the GluD2-derived octapeptide, which is in agreement with previous findings using high-throughput assays.