Periaxin and AHNAK Nucleoprotein 2 Form Intertwined Homodimers through Domain Swapping*

Background: Periaxin and AHNAK nucleoprotein 2 contain a poorly conserved PDZ domain. Results: The crystal structures for the PDZ domains were determined. Conclusion: Both PDZ domains form intertwined domain-swapped homodimers. Significance: The structures have implications for the organization of complexes involving the periaxin/AHNAK family. Periaxin (PRX) is an abundant protein in the peripheral nervous system, with an important role in myelination. PRX participates in large molecular complexes, most likely through the interactions of its N-terminal PSD-95/Discs-large/ZO-1 (PDZ)-like domain. We present the crystal structures of the PDZ-like domains from PRX and its homologue AHNAK nucleoprotein 2 (AHNAK2). The unique intertwined, domain-swapped dimers provide a structural basis for the homodimerization of both proteins. The core of the homodimer is formed by a 6-stranded antiparallel β sheet, with every other strand from a different chain. The AHNAK2 PDZ domain structure contains a bound class III ligand peptide. The binding pocket is preformed, and the peptide-PDZ interactions have unique aspects, including two salt bridges and weak recognition of the peptide C terminus. Tight homodimerization may be central to the scaffolding functions of PRX and AHNAK2 in molecular complexes linking the extracellular matrix to the cytoskeletal network.

The PDZ (PSD-95/Discs-large/ZO-1) domain is one of the most common modular interaction domains, mediating protein-protein interactions by binding to the C terminus, or to an internal sequence, of a target protein. PDZ domains consist of 80 -90 amino acid residues and normally fold into six ␤ strands and two ␣ helices. Peptide ligands bind into a pocket lined by helix ␣2, strand ␤2, and the ␤1-␤2 loop, leading to the formation of an antiparallel ␤ sheet between the ligand and the PDZ domain (1)(2)(3). Usually, the C-terminal carboxylate group of the ligand interacts directly with the backbone of the ␤1-␤2 loop, containing a conserved X⌽G⌽ sequence (also called the GLGF motif); X denotes any amino acid and ⌽ is a hydrophobic residue. Based on binding specificity, PDZ domains can be divided into 3 classes; class I PDZ domains bind to the C-terminal sequence (S/T)-X-⌽-COOH, class II PDZ domains to ⌽-X-⌽-COOH, and class III PDZ domains to (D/E)-X-⌽-COOH (4).
Periaxin (PRX) 2 plays a significant role in myelination of the peripheral nervous system (5,6), comprising 16% of peripheral nervous system myelin protein by weight (7). Genetic defects in PRX result in demyelinating peripheral neuropathies, such as Charcot-Marie-Tooth and Dejerine-Sottas diseases, indicating a crucial role for PRX in the normal development of the peripheral nervous system (8 -10). PRX is also a member of cytoskeletal complexes in lens fibers, and it is considered to function in maturation, packing, and membrane organization of lens fiber cells (11).
Two major isoforms of PRX, L-PRX and S-PRX, of 1461 and 147 amino acids, respectively, are expressed by myelinating Schwann cells. During myelination, L-PRX is first localized in the adaxonal plasma membrane and later, in the abaxonal plasma membrane (5,6). S-PRX is uniformly distributed in the cytoplasm and the nucleus of the Schwann cell, and a function for S-PRX in regulating mRNA splicing has been suggested (12).
Both PRX isoforms share their 127 N-terminal residues, including a predicted PDZ domain (13). The function of the PRX PDZ domain is unknown, but it is essential for PRX dimerization (14). Through its interaction with dystrophin-related protein 2 (Drp2), L-PRX is a member of the periaxin-Drp2-dystroglycan complex. The PRX PDZ domain is apparently not required in this interaction (14). Except for Drp2, no other binding partners for PRX have been reported, and the ligand of the PRX PDZ domain is, hence, also unknown.
The homology of PRX to other proteins is very low; essentially, the only conserved domain is the PDZ domain (15). Even this domain is poorly conserved, and the only homologues with sequence identity Ͼ30% in this region are the giant AHNAK proteins. The PDZ domain of PRX has the highest sequence identity, 57%, with AHNAK2. PRX and the AHNAK proteins form a unique subfamily of PDZ proteins that are likely to have similar functions in complexes linking the extracellular matrix to the cytoskeleton (12).
AHNAKs are giant proteins (molecular mass Ͼ600 kDa) expressed in all muscular cells (16). AHNAK1 (also called desmoyokin) is involved in cytoarchitecture and calcium signaling by directly interacting with several proteins, such as dysferlin, S100B, and calpain3 (17)(18)(19). It is assumed that AHNAK2 localizes at similar sites as AHNAK1 and has similar functions (20). Both AHNAK1 and AHNAK2 can be divided into 3 regions, i.e. an N-terminal PDZ-like region and a C-terminal region with a nuclear localization signal, separated by a large central repeat region (21). Dimerization of AHNAK1 has been reported (12). For both AHNAK1 and PRX, splicing produces two isoforms, i.e. L-AHNAK1/S-AHNAK1 and L-PRX/S-PRX (12).
Here, we report the first crystal structures for PRX and AHNAK2. Their PDZ domains exhibit uniquely intertwined dimers, with extensive three-dimensional domain swapping. Longer constructs of PRX were also characterized in solution and observed to be mainly disordered outside the PDZ domain. The structures of the PRX and AHNAK2 PDZ domains implicate an intriguing mechanism for the formation of stable homodimers. Based on strong interactions in the crystal state, similar dimerization of both proteins is likely to occur also in vivo.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-The production of recombinant PRX PDZ domain variants has been described (22). Briefly, the constructs were expressed as His-tagged variants in Escherichia coli and purified using nickel affinity chromatography. The affinity tag was removed, and the final purification step consisted of size exclusion chromatography. The constructs that were produced include residue ranges 1-147 (full-length S-PRX), 1-127 (the region common to S-and L-PRX), 14 -127, 14 -117, and 14 -107.
A codon-optimized synthetic gene of human AHNAK2 (residues 108 -203) (accession number NM_138420; locus tag NM_138420), encoding a hexahistidine tag and a tobacco etch virus protease cleavage site at the N terminus of the predicted AHNAK2 PDZ domain, subcloned into pETM11, was purchased from MWG Eurofins. The protein was overexpressed in E. coli Rosetta (DE3) cells in ZYM-5052 autoinduction medium (23), containing 50 g/ml of kanamycin and 34 g/ml of chloramphenicol, at 37°C for 7 h. Protein purification was done as for the PRX PDZ domain (22).
Small-angle X-ray Scattering-Synchrotron small-angle xray scattering (SAXS) data for the PRX constructs were collected on beamline P12 of EMBL/DESY at PETRA III (Hamburg, Germany). Samples were prepared at 2-20 mg/ml in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl. Data processing and analysis were performed with the ATSAS package (24). PRIMUS (25) was used for processing and GNOM (26) for estimating the distance distribution. DAMMIN (27), DAMMIF (28), and GASBOR (29) were used for ab initio model building with either dummy residues or chain-like assemblies. BUNCH (30) was used to build hybrid models based on the crystal structure and ab initio built N-and C-terminal extensions. Different models were superimposed using SUPCOMB (31). CRYSOL (32) was used to compare the crystal structure with SAXS data. The molecular weight of the samples was calculated by comparing the forward scattering intensity, I(0), to that of freshly prepared BSA.
Crystallography and Structure Determination-Crystallization of the PRX PDZ domain (residues 14 -107) has been described (22). Briefly, crystals were obtained in 30% PEG 2000MME, 0.15 M KBr after 1 day at 4°C. New higher resolution native data were collected at the P13 EMBL/DESY beamline at PETRA III (Hamburg, Germany). A tungsten-derivatized PRX PDZ crystal was prepared by soaking in 5 mM (NH 4 ) 2 WS 4 for 2 days. The preparation of a xenon-derivatized crystal was executed at BESSY (Berlin, Germany). A crystal was picked and incubated in the xenon chamber at 200 p.s.i. for 8 min. Diffraction data for the derivatized crystals were collected on beamline BL14.1 at BESSY.
Crystals of the AHNAK2 PDZ domain were similarly obtained with sitting-drop vapor diffusion using a 35 mg/ml protein stock in 50 mM Tris (pH 7.5), 100 mM NaCl, with a well solution consisting of 10% PEG8000 and 50 mM KH 2 PO 4 . X-ray diffraction data were collected on the EMBL/DESY beamline P13 at PETRA III (Hamburg).
All data were processed using XDS (33). The phasing of PRX, which was solved first, did not work either by molecular replacement or by the bromide single-wavelength anomalous dispersion method. Thus, various derivatization experiments were performed, and data from a crystal soaked with tungsten showed a strong anomalous signal. In addition, crystals incubated in the xenon chamber also exhibited an anomalous signal from xenon. Combining these two datasets with native diffraction data, phasing was successful. Phasing by MIRAS, followed by automatic model building, was done with AutoSol in Phenix (34). The structure solution of the AHNAK2 PDZ domain was done by molecular replacement using the partially refined PRX PDZ structure as a search model in Phaser (35). Model building was done using COOT (36) and refinement using phenix.refine (37). Molprobity (38) was used for structure validation.
Static Light Scattering-The oligomeric state of the PRX and AHNAK2 constructs in solution (50 mM Tris, pH 7.5, 100 mM NaCl) was studied using static light scattering. Samples of the protein variants used for crystal structure determination were run through a size exclusion column with an Ä kta Purifier (GE Healthcare), and refractive index and light scattering were subsequently measured online using the Optilab rEX and miniDAWN TREOS instruments (Wyatt). Data were analyzed with ASTRA software (Wyatt). The run was similarly performed in the presence of 6 M urea.
Circular Dichroism Spectroscopy-Synchrotron radiation circular dichroism spectra were measured on beamline CD1 at ISA, University of Århus (Denmark). The buffer was 20 mM sodium phosphate (pH 7.5). Each sample was scanned from 280 to 170 nm in 100-m quartz cuvettes at a concentration of 1 mg/ml. Each spectrum was measured 3 times, and the corresponding buffer spectrum was subtracted. Deconvolution was carried out on the Dichroweb server (39).
Urea titration of both proteins was done using a Chirascan Plus (Applied Photophysics) instrument, in a 0.5-mm cuvette. PRX (0.6 mg/ml) was in 10 mM Tris (pH 7.5), 20 mM NaCl, and AHNAK2 (0.2 mg/ml) in H 2 O. Spectra were also measured in the presence of up to 6 M urea.
Native PAGE-The native PAGE samples of PRX and AHNAK2 PDZ domains were prepared at different concentrations of SDS and urea in 50 mM Tris (pH 7.5), 100 mM NaCl. The gel was run in 25 mM Tris, 195 mM glycine (pH 8.5) at 4°C for 2 h and Coomassie stained.

Dimerization of PRX and AHNAK2 N-terminal Domains in Solution-
The recombinant PRX PDZ domain was a folded dimer, as evidenced by CD spectroscopy, size exclusion chromatography, and static light scattering (Fig. 1). The PDZ domain of AHNAK2 was also homodimeric in solution (Fig.  1D). CD spectroscopy indicated both proteins were stable in 1 M urea, but started unfolding in 3 M urea (Fig. 1B). Static light scattering proved that the dimers observed in solution were dissociated into unfolded monomers at 6 M urea ( Fig. 1, C and D). Native gel electrophoresis further showed that the mobility of neither protein was affected by urea concentrations up to 1 M (Fig. 1E), whereas changes in mobility were seen with SDS. Hence, the dimers are stable in solution at relatively high concentrations of urea.
Three-dimensional Domain-swapped Dimeric Structure of the PRX PDZ Domain-The crystal structure of the PRX PDZ domain was solved at 2.7-Å resolution ( Table 1). Three monomers exist in one asymmetric unit, and the structure presents unique dimeric folding, involving a high degree of domain swapping and intertwining between the two monomers (Fig. 2).
The PRX PDZ domain displays 5 major ␤ strands and 2 ␣ helices in one monomer (Fig. 2B). Compared with other PDZ domains, which generally have 6 ␤ strands, the PRX PDZ domain does not have separate strands ␤4 and ␤5, but a long ␤4/5 strand, and the ␤4-␤5 loop is missing. The loss of this loop results in the change of direction of ␣2 and ␤6 in the overall monomer structure. These units further intertwine with the neighboring monomer and vice versa, and two monomers form a domain-swapped homodimer (Fig. 2).
Homo-and heterodimerization of PDZ domains have been observed. However, only the ZO-1 PDZ2 domain was reported to be a domain-swapped homodimer (40), whereas the PDZ domains from Shank1 and GRIP1 (glutamate receptor interacting protein 1) form homodimers without domain swapping (41,42). The PRX PDZ domain exhibits a dimerization mode distinct from any other known structure (Fig. 2C). Although in ZO-1 PDZ2, a domain-swapped homodimer is formed through swapping the N-terminal ␤1 and ␤2 strands with the neighboring molecule, much more extensive domain swapping is present in PRX, via the interchange of ␣2 and ␤6 between two monomers (Fig. 2).
In the PRX dimer, a 6-stranded antiparallel ␤ sheet is formed by the two chains, with the two ␤4/5 strands in the center (Fig.  3). Every second ␤ strand comes from a different chain, resulting in a highly intertwined structure for the homodimer. This sheet is formed of strands ␤1, ␤4/5, and ␤6 of the A and B chains, in the order In addition, the ␣2 helices are side by side in the dimer structure.
Between the ␣2 helices from the two chains,stacking is present between the side chains of Phe 75 and Tyr 90 . This stacking also includes Pro 89 and Phe 72 ; in addition, there is a buried hydrophobic core (Fig. 4A). Another unique conformation is present on the ␤1 strand. In PRX, ␤1 ends at Glu 22 , and a bulge is observed from Thr 23 to Ala 25 . Gln 26 -Gly 28 acquire an additional small ␤ strand conformation, ␤1Ј, by backbone hydrogen bonding with Tyr 90 -Val 92 on strand ␤6 of the neighboring monomer. This bulge is formed due to the insertion of one residue into the ␤1 strand in PRX (Fig. 4B). The side chain of Thr 23 makes the missing hydrogen bond to the neighboring strand. The two peptide binding sites of the PRX homodimer are composed between helix ␣2 and strand ␤2 from different chains. Because the first residue of the ␣2 helix is Tyr, the PRX PDZ domain can be categorized into class III and may specifically bind to a C-terminal (D/E)-X-⌽-COOH motif on the ligand protein (43). A backbone peptide group between Val 29 and Ser 30 , expected to recognize the target ligand C terminus, is flipped compared with earlier PDZ domain structures (Fig. 4C). As a result, the carbonyl moiety points toward the expected position of the ligand peptide carboxyl terminus.
As expected from sequence alignments, the PRX sequence 28 GVSGI 32 , homologous to the GLGF motif, is located in the C-terminal carboxylate binding ␤1-␤2 loop (Fig. 4, C and D). In PRX, this loop is 2-6 amino acids shorter than in other PDZ structures. Also loop ␤2-␤3 is shorter than in most other PDZ structures (Fig. 4D). In addition, the C-terminal end of helix ␣2 in PRX leans slightly toward ␤2. Due to these factors, the apparent volume of the peptide binding pocket of the PRX PDZ domain is small compared with other PDZ domains. However, it is possible that the peptide binding pocket opens up when a binding partner enters.
Solution Structure-The solution structures of several constructs of the PRX PDZ domain were modeled based on SAXS data (Fig. 5). The forward scattering intensity indicated quantitative homodimer formation by each construct in solution ( Table 2). The data for PRX14 -107, which was also successfully used for crystallization, suggested compact folding, whereas the longer constructs exhibited more extended structures. The latter was also exemplified by normalized Kratky plots (Fig. 5C) (44).
The crystal structure fit the SAXS data very well (Fig. 5D), indicating highly similar conformations in the crystal state and in solution. The N-and C-terminal extensions gradually increased the dimensions of PRX (Table 2), suggesting they are elongated. Three-dimensional models of the various constructs confirmed the above findings, and N-and C-terminal extensions can be distinguished in the models (Fig. 5, E  and F).
The AHNAK2 PDZ Domain Structure and Peptide Binding-To shed light on the uniqueness of the PRX PDZ domain homodimer structure, we also determined the structure of its closest homologue. The crystal structure of the human AHNAK2 PDZ domain was refined at 1.75-Å resolution, with a dimer in the asymmetric unit ( Table 1). The overall structure of the AHNAK2 PDZ domain displays exactly the same domainswapped homodimer as PRX (Fig. 6, A and B), indicating that PRX and AHNAK2 form a distinct subgroup of the PDZ domain family, with a unique, previously unseen way of intertwined folding. However, strand ␤2 of AHNAK2 is leaning more toward strand ␤3 (Fig. 6A); thus, the peptide binding pocket is larger than in PRX.
A fortuitous feature in the AHNAK2 structure is the binding of the C terminus of a crystallographic symmetry mate in one, but not the other, peptide binding pocket of the homodimer (Fig. 6C); hence, both the unliganded and liganded binding sites were trapped in the same crystal. The sequence of the bound C terminus is Glu-Glu-Trp-Ala-COOH (residues 200 -203). Thus, the AHNAK2 PDZ domain can be categorized into class III, as also expected from its sequence. Three backbone hydrogen bonds are present between strand ␤2 and the ligand (Fig. 6C). In addition, several side chain hydrogen bonds are present (Fig. 6C). An interesting conformation in the peptide binding site of AHNAK2 is found in the peptide bond between Ala 123 and Ser 124 . In most other peptide-bound PDZ domain structures, the corresponding peptide bond is oriented so that the NH group interacts with the C-terminal carboxylate of the ligand. This interaction is missing in AHNAK2 (Fig. 6, C and E). The same backbone conformation is also found in the empty peptide binding pocket, as well as in the unliganded PRX PDZ domain (Fig. 4C). Thus, this unexpected conformation is a common property of these homologous PDZ domains, and it is not affected by ligand binding. This segment is in the ␤1-␤2 loop, at the conserved GLGF motif (GVSGI in PRX, GASGY in AHNAK2) for carboxylate binding. The Ser side chain in this motif in AHNAK2 makes a hydrogen bond to the ligand C terminus (Fig. 6C); this residue is also conserved in PRX and may be involved in ligand recognition by both proteins.
To investigate conformational changes induced by ligand peptide binding, the structures of the two AHNAK2 monomers were superimposed (Fig. 6B). The two binding sites are nearly identical; some differences are found in temperature factors. The peptide-bound pocket, formed of strand ␤2 from chain A and helix ␣2 from chain B, has lower B factors than the empty pocket (Fig. 6D). The ligand-PDZ domain interaction decreases the dynamics of ␣2, ␤2, and ␤3, although does not affect the binding site conformation.

DISCUSSION
Both PRX and the AHNAK proteins participate in large protein complexes at the plasma membrane, linking the extracellular matrix to the cytoskeleton. Although evidence exists for dimerization in these proteins (12,14), the molecular details have been missing. We provide here a structural basis for the dimeric assembly of PRX/AHNAK, and the surprising inter- twined folding of these two PDZ domains is likely to be relevant to the overall architecture and stability of the respective multiprotein complexes, including the EPPD (ezrin, periplakin, periaxin, and desmoyokin/AHNAK)) complex in lens fibers and the dystrophin glycoprotein complex in Schwann cells.
It has been assumed that the PRX PDZ domain is canonical (45,46). A new category of homodimeric PDZ domain struc-tures is now provided by PRX and AHNAK2, which have a distinct domain-swapped fold compared with other known PDZ domain structures. By default, the dimerization also dictates that there are two peptide binding sites, with opposite orientations, next to each other in this PDZ assembly, which may be relevant for building large ordered protein complexes involving PRX/AHNAK. Experimental evidence has been presented for PDZ domain-mediated dimerization of both proteins before; PRX dimerization was shown by yeast two-hydrid experiments, pulldowns from nerve lysates, affinity chromatography, and co-immunoprecipitations from mammalian cells overexpressing PRX constructs (14). On the other hand, AHNAK was previously shown to form detergent-resistant  homo-and heterodimers (between S-and L-AHNAK) (12). Hence, the tight homodimers observed here are likely to be of relevance in a cellular environment.
Despite the low homology to even the closest relatives (Fig.  7A), a comparison of sequences pinpoints unique features of PRX and AHNAK2, which correlate with their structures. The insertion of one residue into strand ␤1 in both proteins is related to the formation of a bulge and division of ␤1 into two short strands. On the other hand, most PDZ domains have a glycine residue in the ␤4 -␤5 turn; in PRX and AHNAK2, this residue is deleted, and a long continuous ␤4/5 strand is formed. In general, shortening hinge loops in domainswapped proteins tends to lead to oligomerization (47,48), and making such loops longer may generate monomers instead of domain-swapped dimers (48,49). The folding pathways of PDZ domains have been studied before, and strands ␤1, ␤4, and ␤6 were shown to comprise a folding nucleus for the PTP-BL second PDZ domain (50). The corresponding strands in PRX and AHNAK2 form the 6-stranded antiparallel ␤ sheet between the two intertwined chains. It is possible that interactions within this ␤ sheet act as determinants for homodimeric folding.
Although limited data exist on domain swapping in PDZ domains, it is relatively common in the protein universe. Intertwined structures, on the other hand, are much more rare; domain swapping normally would involve the swap of a single secondary structure element. One example of an intertwined domain-swapped protein is the dimeric Ig domain of RelB (51). Usually, domain swapping involves secondary structure elements at the N or C terminus of the protein (48), and the case of

Intertwined Dimerization of PDZ Domains
PRX/AHNAK2 is unique, due to the fact that the two chains for a large part wrap around each other. The protein-ligand backbone interactions in the liganded AHNAK2 structure are weaker than in other liganded PDZ structures, i.e. only 3 hydrogen bonds are present, whereas in general, more than 4 hydrogen bonds exist in other complexes. The conformation of the carboxylate-binding loop in PRX/ AHNAK2 suggests it may not be optimal for recognizing the C terminus of a ligand, because a backbone carbonyl group points toward the ligand carboxyl group. It is possible that this arrangement is more suitable for internal peptide motif binding, in which case the flipped carbonyl group could interact with a backbone amide from a ligand. The available structures of non-C-terminal peptide complexes of PDZ domains (Fig.  7B), on the other hand, each imply different mechanisms of recognition (52)(53)(54). It should be mentioned, however, that structural information on PDZ domains binding non-C-terminal sequences is very scarce.
PRX and AHNAK2 are likely to regulate the organization of multimolecular complexes. The liganded AHNAK2 PDZ structure shows that the PDZ domains with this intertwined homodimeric folding can interact with target sequences similarly to canonical PDZ domains. Intriguingly, the volume of the peptide binding pocket in PRX and AHNAK2 differs 2-fold, whereas the corresponding volume for a canonical PDZ domain lies between these two (Fig. 7C). The fact that both binding sites in the AHNAK2 dimer are very similar suggests that no large-scale changes occur upon peptide binding; hence, it is possible that the binding determinants for PRX and AHNAK2 are different. Ligands for these PDZ domains have not been discovered yet. The PRX and AHNAK2 PDZ domain structures provide starting points in the search for their binding partners. Likely possibilities include recognition of class III target sequences or internal peptide motifs, or combinations of both.
The significance of the homodimerization of PRX and AHNAK2 may lie in assembling the matrix lining the intracellular side of membrane; i.e. the PRX basic domain binds to the Drp2 spectrin-like domain. Then, the spectrin-like, WW, and ZZ domains of Drp2 interact with other proteins, such as dystroglycan, utrophin, or Dp116, to make a large complex. The stable homodimerization of two domain-swapped PDZ monomers certainly would be one of the secure points in such a large complex, and the symmetry of the dimer would assist to arrange the complex. A corresponding scenario is likely to occur for AHNAKs, which are involved in similar complexes (20,55). In fact, both PRX and AHNAK are members of the EPPD complex in lens fiber cells (56), where they may even function together. Largest differences are seen in helix ␣2 from one chain and strands ␤2 and ␤3 from the other chain (arrows). E, the peptide binding site between ␣2 and ␤2 in AHNAK2 (pink) and PAR-6 (green) (59). Note the distinct conformations of the ␤1-␤2 loop. The bound peptide ligands are shown in orange and gray, respectively, and the hydrogen bonds between the terminal carboxyl group and backbone amide groups as dashed lines (AHNAK-2, magenta; PAR-6, green). The arrow points to the carbonyl group of Ala 123 .
Interestingly, PRX is also required for the hexagonal packing of lens fibers (11). The domain organization of PRX and AHNAKs, as well as the structures of their dimerization domains, may be signs of these proteins being involved in macromolecular complexes with similar assembly pathways (57).
Taken together, the structures of PRX and AHNAK2 PDZ domains reveal unique folding properties, with extensive three-dimensional domain swapping in an intertwined manner. Our data provide insights into identifying binding partners for these domains, which are unknown to date, and thus, will FIGURE 7. Comparison of PRX and AHNAK2 sequence and binding cavity. A, structural sequence alignment of PRX and AHNAK2 PDZ domains with two of their closest sequence homologues in PDB, the human SYNJ2BP (Protein Data Bank entry 2JIN) and human ZO-1 PDZ3 (Protein Data Bank entry 3TSV) (58). The secondary structures of the PDZ domains from AHNAK2 and SYNJ2BP are shown above and below the alignment, respectively. The image was prepared using Espript (60). The positions of the insertion in ␤1 (green) and the deletion in ␤4/5 (red) are indicated with triangles above the alignment. B, structures of internal peptide-binding PDZ domains. The PDZ domains are colored cyan, the binding ligands yellow, and the hydrogen bonds green. Note the varying conformations of the ␤1-␤2 loop. Left, the PAR-6 PDZ domain with the PALS1 peptide (53). Instead of the terminal carboxylate of P 0 , the side chain and backbone carbonyl of Asp(P ϩ1 ) form hydrogen bonds with the GLGF loop. Middle, ␣-1 syntropin PDZ domain with neuronal nitric-oxide synthase (52). The carbonyl group of P 0 interacts with two backbone nitrogen atoms. Right, the Dishevelled-2 PDZ domain with N1 inhibitory peptide (54). The backbone conformation of P 0 of the ligand is unique. The carboxylate in the Asp(P ϩ1 ) side chain is located at a position corresponding to a canonical peptide C terminus, interacting with the GLGF loop. C, estimation of peptide ligand binding pocket volumes of AHNAK2 (red), PRX (blue), and SYNJ2BP (magenta) using POVME (61). The pocket volumes were 912, 576, and 1200 Å 3 , respectively. The liganded and unliganded pockets in the AHNAK2 homodimer are essentially identical, excluding large scale induced fitting of the peptide.