Sequence-specific 1H, 13C, and 15N assignment of the extended PDZ3 domain of the protein tyrosine phosphatase basophil-like PTP-BL

Protein tyrosine phosphatase basophil-like (PTP-BL), also known as PTPN13, represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the complete resonance assignments of the extended PDZ3 domain of PTP-BL. These assignments provide a basis for the detailed structural investigation of the interaction between the PDZ domains of PTP-BL as well as of their interaction with ligands. It will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTB-BL.     

Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms

PDZ domains most commonly bind the C‐terminus of their protein targets. Typically the C‐terminal four residues of the protein target are considered as the binding motif, particularly the C‐terminal residue (P0) and third‐last residue (P‐2) that form the major contacts with the PDZ domain's “binding groove”. We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C‐terminal P0 residue bound in the binding groove. Importantly, in some cases, the P‐2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side‐chains and the αB helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical “perpendicular” binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.     

Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-A resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at -1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at -3 and -5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved beta B/beta C loop and N-terminal beta A strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal beta A strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.     

The binding of the PDZ tandem of syntenin to target proteins

PDZ domains are among the most abundant protein modules in the known genomes. Their main function is to provide scaffolds for membrane-associated protein complexes by binding to the cytosolic, C-terminal fragments of receptors, channels, and other integral membrane proteins. Here, using both heteronuclear NMR and single crystal X-ray diffraction, we show how peptides with different sequences, including those corresponding to the C-termini of syndecan, neurexin, and ephrin B, can simultaneously bind to both PDZ domains of the scaffolding protein syntenin. The PDZ2 domain binds these peptides in the canonical fashion, and an induced fit mechanism allows for the accommodation of a range of side chains in the P(0) and P(-)(2) positions. However, binding to the PDZ1 domain requires that the target peptide assume a noncanonical conformation. These data help explain how syntenin, and perhaps other PDZ-containing proteins, may preferentially bind to dimeric and clustered targets, and provide a mechanistic explanation for the previously reported cooperative ligand binding by syntenin's two PDZ domains.     

Design, synthesis, and evaluation of linear and cyclic peptide ligands for PDZ10 of the multi-PDZ domain protein MUPP1

PDZ10 is the 10th of 13 PDZ domains found within MUPP1, a cytoplasmic scaffolding protein first identified as an endogenous binding partner of serotonin receptor type 2c (5HT2c). This association, as with those of several other interacting proteins that have subsequently been identified, is mediated through the C-terminal tail of the PDZ domain partner. Using isothermal titration calorimetry (ITC), we measured the thermodynamic binding parameters [changes in Gibbs free energy (DeltaG), enthalpy (DeltaH) and entropy (TDeltaS)] of the isolated PDZ10 domain for variable-length N-acetylated peptides from the 5HT2c serotonin receptor C-terminal sequence, as well as for octapeptides of eight other putative partner proteins of PDZ10 (5HT2a, hc-kit, hTapp1, mTapp2, TARP, NG2, claudin-1, and HPV-18 E6). In length dependence studies of the 5HT2c sequence, the maximal affinity of the peptides leveled off rapidly and further elongation did not significantly improve the dissociation constant (Kd) of 11 microM observed with the pentapeptide. Among the native partners of PDZ10, octapeptides derived from the hc-kit and 5HT2c proteins were the strongest binders, with Kd values of 5.2 and 8.5 microM, respectively. The heat capacity change (DeltaCp) for the 5HT2c octapeptide was determined to be -94 cal/mol, and a calculated estimate indicates burial of polar and apolar surface areas in equal measure upon ligand binding. Peptides with phosphoserine at either the P-1 or P-2 position experienced decreased affinity, which is in accord with the hypothesis that reversible phosphorylation is a possible mechanism for regulating PDZ domain-mediated interactions. Additionally, two conformationally constrained side chain-bridged cyclic peptide ligands were also designed, prepared, evaluated by ITC, and shown to bind PDZ10 primarily through a favorable change in entropy.     

Sequence-specific 1H, 13C, and 15N backbone assignment of the 28 kDa PDZ2/PDZ3 tandem domain of the protein tyrosine phosphatase PTP-BL

Protein tyrosine phosphatase-basophil like (PTP-BL) represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the backbone assignments of the PDZ2/PDZ3 tandem domain of PTP-BL. These assignments now provide a basis for the detailed structural investigation of the interaction between the PDZ domains 2 and 3 of PTP-BL. It will lead to a better understanding of the proposed scaffolding function of this tandem domain in multi-protein complexes assembled by PTB-BL. Christian P. Fetzer, Janelle Sauvageau and Gerd Kock contributed equally to this work.     

Crystal structure of GRIP1 PDZ6-peptide complex reveals the structural basis for class II PDZ target recognition and PDZ domain-mediated multimerization

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. Glutamate receptor-interacting protein (GRIP)/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-alpha. In addition the PDZ456 domain mediates the formation of homo- and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-alpha at resolutions of 1.5 and 1.8 A, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at alphaB5 rather than conserved Leu-732 at alphaB1 makes a direct hydrophobic contact with the side chain of the Tyr at the -2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix alphaB, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.     

Structural determinants of the Na+/H+ exchanger regulatory factor interaction with the beta 2 adrenergic and platelet-derived growth factor receptors

The Na(+)/H(+) exchanger regulatory factor (NHERF) binds through its PDZ1 domain to the carboxyl-terminal sequences NDSLL and EDSFL of the beta(2) adrenergic receptor (beta(2)AR) and platelet-derived growth factor receptor, respectively, and plays a critical role in the membrane localization and physiological regulation of these receptors. The crystal structures of the human NHERF PDZ1 domain bound to the sequences NDSLL and EDSFL have been determined at 1.9- and 2.2-A resolution, respectively. The beta(2)AR and platelet-derived growth factor receptor ligands insert into the PDZ1 binding pocket by a beta-sheet augmentation process and are stabilized by largely similar networks of hydrogen bonds and hydrophobic contacts. In the PDZ1-beta(2)AR complex, the side chain of asparagine at position -4 in the beta(2)AR peptide forms two additional hydrogen bonds with Gly(30) of PDZ1, which contribute to the higher affinity of this interaction. Remarkably, both complexes are further stabilized by hydrophobic interactions involving the side chains of the penultimate amino acids of the peptide ligands, whereas the PDZ1 residues Asn(22) and Glu(43) undergo conformational changes to accommodate these side chains. These results provide structural insights into the mechanisms by which different side chains at the position -1 of peptide ligands interact with PDZ domains and contribute to the affinity of the PDZ-ligand interaction.     

Suppression of hepatitis B viral gene expression by protein-tyrosine phosphatase PTPN3

Summary Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of ³⁵⁹serine and ⁸³⁵serine of 14-3-3β binding sites to alanine, which slightly reduces the interaction with 14-3-3β, does not influence the PTPN3 effect. In contrast, mutation of the invariant ⁸⁴²cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3β, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3β, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression. En-Chi Hsu, Yen-Cheng Lin have equal contributions to this work.     

Structural insights into the PIP2 recognition by syntenin-1 PDZ domain

Lipid-mediated regulatory mechanism of the C-terminal ligand binding to PDZ domains is not fully understood, despite their roles in subcellular organization. Here, we provide structural insights into the phosphatidylinositol 4,5-bisphosphate (PIP₂) recognition mode of a PDZ domain, as revealed from the crystal structure of the phosphate-bound PDZ domain. Two adjacent phosphate ions bind to the basic residues close to the amino terminus of the α2 helix in the Tamalin PDZ domain, reflecting an interaction mode of the two phosphate groups of PIP₂. Based on the observed location of the two phosphate molecules within the PDZ domain, we built the docking model of PIP₂ with the PDZ domain of the well-known PIP₂-binding protein, syntenin-1. This model suggests that the hydrophobic diacylglycerol group of PIP₂ could contact the ligand-binding groove of the PDZ domain. These structural features well explain biological phenomena, which were previously reported for the PIP₂-mediated PDZ ligand-binding regulation.     

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.     

Energetic determinants of internal motif recognition by PDZ domains

PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.     

The second PDZ domain of INAD is a type I domain involved in binding to eye protein kinase C. Mutational analysis and naturally occurring variants

INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cbeta(4)), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile(-3) in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second alpha-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaD contains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction.     

Tandem PDZ repeats in glutamate receptor-interacting proteins have a novel mode of PDZ domain-mediated target binding

The interaction of the glutamate receptor subunits 2 and 3 (GluR2/3) with multi-PDZ domain glutamate receptor-interacting protein (GRIP) is important for the synaptic trafficking and clustering of the receptors. Binding of GluR2/3 to GRIP requires both the fourth and fifth PDZ domains (PDZ4 and PDZ5) to be covalently linked, although only one PDZ domain is directly involved in binding to the receptor tail. To elucidate the molecular basis of this mode of PDZ domain-mediated target recognition, we solved the solution structures of the PDZ45 tandem and the isolated PDZ4 of GRIP. The two PDZ domains form a compact structure with a fixed interdomain orientation. The interdomain packing and the stable folding of both PDZ domains require a short stretch of amino acids N-terminal to PDZ4 and a conserved linker connecting PDZ4 and PDZ5. PDZ4 contains a deformed aB-bB groove that is unlikely to bind to carboxyl peptides. Instead, the domain stabilizes the structure of PDZ5.     

Degradation of tyrosine phosphatase PTPN3 (PTPH1) by association with oncogenic human papillomavirus E6 proteins

Oncoproteins from DNA tumor viruses associate with critical cellular proteins to regulate cell proliferation, survival, and differentiation.Human papillomavirus (HPV) E6 oncoproteins have been previously shown to associate with a cellular HECT domain ubiquitin ligase termed E6AP (UBE3A). Here we show that the E6-E6AP complex associates with and targets the degradation of the protein tyrosine phosphatase PTPN3 (PTPH1) in vitro and in living cells. PTPN3 is a membrane-associated tyrosine phosphatase with FERM, PDZ, and PTP domains previously implicated in regulating tyrosine phosphorylation of growth factor receptors and p97 VCP (valosin-containing protein, termed Cdc48 in Saccharomyces cerevisiae) and is mutated in a subset of colon cancers. Degradation of PTPN3 by E6 requires E6AP, the proteasome, and an interaction between the carboxy terminus of E6 and the PDZ domain of PTPN3. In transduced keratinocytes, E6 confers reduced growth factor requirements, a function that requires the PDZ ligand of E6 and that can in part be replicated by inhibiting the expression of PTPN3. This report demonstrates the potential of E6 to regulate phosphotyrosine metabolism through the targeted degradation of a tyrosine phosphatase.     

Solution structure of AF-6 PDZ domain and its interaction with the C-terminal peptides from Neurexin and Bcr

AF-6 is a key molecule essential for structure organization of cell-cell junction of polarized epithelia. It belongs to a novel cell-cell adhesion system. The AF-6 PDZ domain mediates interactions by binding to a specific amino acid sequence in target proteins. Here we report the solution structure of the AF-6 PDZ domain determined by NMR. Previously, the AF-6 PDZ domain was considered to be a class II PDZ domain. However we found that a unique hydrophilic amino acid, Gln70, at position alphaB1 makes the alphaB/betaB groove of the AF-6 PDZ domain significantly different from that of the canonical class II PDZ domain. The AF-6 PDZ domain does not have the second hydrophobic binding pocket, and the N-terminal end of alphaB is closer to betaB. Using BIACORE and NMR chemical shift perturbation experiments, we have studied the binding characteristics of the PDZ domain to the C-terminal peptide of Neurexin, KKNKDKEYYV, and that of Bcr, KRQSILFSTEV. The C-terminal peptide of Neurexin is a class II ligand, whereas that of Bcr is a class I ligand. The dissociation constants of these ligands were 4.08 x 10(-7) and 2.23 x 10(-6) m, respectively. Each of the four C-terminal positions in Neurexin and Bcr may contribute to the interaction. The three-dimensional models of the AF-6 PDZ-Neurexin C-terminal peptide complex and the AF-6 PDZ-Bcr C-terminal peptide complex were built up by molecular dynamics simulations. Unlike the canonical class II PDZ domain, Ala74 at alphaB5 rather than the residue at alphaB1 makes direct hydrophobic contact with the side chain of Tyr at the -2 position of the ligand.     

An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for understanding the interaction networks they establish. We determined the binding preference of the five PDZ domains in the protein tyrosine phosphatase PTP-BL by screening a random C-terminal peptide lambda phage display library. Interestingly, the potential of PDZ2 to interact with class III-type ligands was found to be modulated by the presence of PDZ1. Structural studies revealed a direct and specific interaction of PDZ1 with a surface on PDZ2 that is opposite the peptide binding groove. Long-range allosteric effects that cause structural changes in the PDZ2 peptide binding groove thus explain the altered PDZ2 binding preference. Our results experimentally corroborate that the molecular embedding of PDZ domains is an important determinant of their ligand binding specificity.     

Solution structure of the PDZ2 domain from cytosolic human phosphatase hPTP1E complexed with a peptide reveals contribution of the beta2-beta3 loop to PDZ domain-ligand interactions

The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.     

Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.