Structure of an L27 Domain Heterotrimer from Cell Polarity Complex Patj/Pals1/Mals2 Reveals Mutually Independent L27 Domain Assembly Mode*

Background: Tandem L27 domains are important for multidomain proteins to assemble into supramolecular complexes for cell polarity regulation. Results: Tandem L27 domain-mediated tripartite Patj/Pals1/Mals2 and DLG1/CASK/Mals2 complexes form in a mutually independent assembly mode. Conclusion: The mutually independent assembly mode may be a novel mechanism for tandem L27 domain-mediated, tripartite complex formation. Significance: These findings reveal the distinct mechanism of tandem L27 domain-mediated assembly of obligate supramolecular complexes. The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode.

Establishment of cell polarity is indispensable for most eukaryotic cell functions. The asymmetric distribution of a set of evolutionarily conserved cell polarity proteins or lipids is required for establishing and maintaining cell polarization (1,2). Many of these conserved proteins are multidomain scaffold proteins, and they often interact with each other to assemble supramolecular protein complexes, thereby mediating the fundamental process for cell polarity (3,4). The L27 domain, initially identified in the Caenorhabditis elegans Lin-2 and Lin-7 proteins (5), exists in many scaffold proteins and is involved in assembling essential supramolecular protein complexes that play critical roles in cell polarity (6 -11).
The structural basis of cognate L27 domain heterodimer assembly has been studied extensively by NMR and x-ray crystallography and has previously led to the proposal of a unified model of symmetric L27 homotetramers (dimer of heterodimers) (17)(18)(19)(20). Recently, the structure of a tandem L27 domain-mediated tripartite L27 DLG1 /(L27N-L27C) MPP7 / L27 Mals3 complex showed the asymmetric, cooperative assembly of a heterotrimer consisting of two cognate pairs of heterodimeric L27 domains (21).
In this study, we solved the crystal structure of the L27 domain heterotrimer from the tripartite complex Patj/Pals1/ Mals2. The structure of the L27 domain complex, together with data derived from various biochemical studies, establishes a novel, symmetric, and mutually independent assembly mode of heterotrimer formation mediated by tandem L27 domains in the specific tripartite Patj/Pals1/Mals2 complex. Additionally, we showed that another specific tripartite DLG1/CASK/Mals2 complex is also formed via tandem L27 domain-mediated, symmetric, and mutually independent assembly.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-To construct a singlechain fusion protein of the L27 Patj /(L27N-L27C) Pals1 /L27 Mals2 complex (supplemental Fig. S1A and molecule 1 in supplemental Fig. S1C), DNA fragments corresponding to the L27 domain of rat Patj (residues 1-68), the tandem L27 domains of human Pals1 (residues 119 -232), and the L27 domain of mouse Mals2 (residues 3-66) were amplified by PCR and linked with two rhinovirus 3C protease-cleavable segments (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro), and a triglycine cassette (GGG) was inserted before the second 3C proteasecleavable segment. The single open reading frame was cloned into the pET32a vector (Novagen, San Diego, CA), in which the S-tag and the thrombin recognition site were replaced with a sequence encoding a tobacco etch virus (TEV) proteasecleavable segment (Glu-Asn-Leu-Tyr-Phe-Gln-Ser). BL21(DE3) CodonPlus Escherichia coli cells harboring the expression plasmid were grown in LB medium at 37°C until the A 600 reached 0.6 and then induced with 0.3 mM isopropyl-␤-Dthiogalactoside at 16°C for ϳ16 -18 h. After centrifugation at 5000 rpm for 15 min, E. coli cells were resuspended in T 50 N 500 I 5 buffer (50 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 5 mM imidazole) supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 1 g/ml antipain. The cells were then lysed by sonication. After the lysates had been centrifuged at 18,000 rpm for 30 min, the supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen, Valencia, CA) that was equilibrated with T 50 N 500 I 5 buffer. The nickel-nitrilotriacetic acid column was washed with 3 column volumes of T 50 N 500 I 5 buffer. The His 6 -tagged protein was eluted with T 50 N 500 I 5 buffer containing 500 mM imidazole. The eluted proteins were digested with TEV protease overnight to remove the N-terminal His 6 tag and then loaded onto a HiLoad 26/60 Superdex 200 size-exclusion column (GE Healthcare) and eluted with T 50 N 50 E 1 D 1 buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, and 1 mM DTT) at a flow rate of 2.5 ml/min. Each fraction of the column eluate was 5 ml. The single-chain fusion protein peak of the L27 Patj /(L27N-L27C) Pals1 / L27 Mals2 complex was identified by SDS-PAGE, and the corresponding fractions were pooled and concentrated to 90 mg/ml for crystallization trials. To obtain the three separate chains of the L27 Patj /(L27N-L27C) Pals1 /L27 Mals2 complex (supplemental Fig. S2A), 3C protease was added to the single-chain fusion protein to cleave the covalent linker. A Se-Met-substituted version of the L27 Patj /(L27N-L27C) Pals1 /L27 Mals2 single-chain fusion protein was produced following the same protocol that was used for the wild-type protein, with the exception that methionine auxotroph E. coli B834 (DE3) cells and LeMaster medium were used to express the recombinant protein.
Analytical Ultracentrifugation-Sedimentation velocity (SV) and sedimentation equilibrium (SE) experiments were performed in a Beckman Coulter XL-I analytical ultracentrifuge (Beckman Coulter) using double-sector or six-channel centerpieces and sapphirine windows. Before the experiments, the proteins were transferred to buffer containing 50 mM PBS, pH 7.4, 100 mM NaCl, and 1 mM EDTA by Superose 12 10/300 GL column (GE Healthcare). Proteins at absorbances of 0.6 and 1.2 at 280 nm were loaded into double-sector cells for SV experiments, which were conducted at 42,000 rpm and 10°C and with absorbance detected at 280 nm. For SE experiments, data were collected at 4,000, 22,000, and 27,000 rpm and 4°C by interference detection using six-channel cells. The concentrations of the proteins were ϳ27 and 40 M. The buffer composition (density and viscosity) and protein partial specific volume (V-bar) were obtained sing the SEDNTERP program (available through the Boston Biomedical Research Institute). The SV and SE data were analyzed using the SEDFIT and SEDPHAT programs (22,23), respectively.
Crystallization and Data Collection-The wild-type L27 Patj / (L27N-L27C) Pals1 /L27 Mals2 protein complex in the single-chain fusion form (90 mg/ml in T 50 N 50 E 1 D 1 buffer) was crystallized using sitting drop vapor diffusion equilibrated with a reservoir solution of 2.8 M sodium acetate trihydrate, pH 7.0. Crystals were grown over 1 month at 20°C and directly flash-frozen in liquid nitrogen. The Se-Met-substituted crystals were produced in the same manner as the wild-type crystals. Diffraction data sets were collected using beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) and processed using the HKL2000 software (24). Both wild-type and Se-Met-substituted crystals belonged to the space group P6 1  Structure Determination and Refinement-The HKL2MAP program (25) was used to search eight selenium sites in one asymmetric unit cell. The initial phases were then calculated by PHENIX software (26). Model building and refinement were performed using COOT (27) and PHENIX (28). After the initial main-chain model was built, the wild-type data were applied to carry out iterative refinement to assign all side chains. The final structure had an R cryst value of 18.0% and an R free value of 22.1%. The Ramachandran plot generated by the program PROCHECK (29) shows that 95.0% residues are in their most favored regions, 4.6% residues are in additional allowed regions, 0.4% residues are in generously allowed regions, and no residue is in disallowed regions. Detailed data collection and refinement statistics are summarized in supplemental Table S1. All figures were made with the PyMOL program (30).
Glutathione S-Transferase(GST) Pulldown Assays-GST-L27 Patj , GST-Mals2, and GST-Pals1 fusion proteins were expressed in E. coli BL21 (DE3) CodonPlus cells and purified using a glutathione-Sepharose 4B column (GE Healthcare) and a Superdex 200 size-exclusion column. Transfected HEK293T cells were lysed using 500 l of ice-cold cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 1 g/ml antipain) and cleared by centrifugation at 13,000 rpm for 20 min at 4°C. Soluble fractions were incubated with GST fusion proteins at 4°C for 2 h. Glutathione-Sepharose 4B beads (GE Healthcare) were then added for further incubation at 4°C for 2 h. The beads were washed with cell lysis buffer and boiled in SDS sample buffer. The prepared samples were separated by SDS-PAGE and analyzed using Western blots.
Co-immunoprecipitation-HEK293T cells were plated at ϳ6.5 ϫ 10 6 cells in 10-cm dishes. The following day, 70 -80% confluent cells were transfected with the indicated amounts and various combinations of plasmids. At 24 h after transfection, HEK293T cells were lysed and cleared by centrifugation at 13,000 rpm for 20 min at 4°C. The supernatants were then incubated with anti-GFP antibody (1 g) at 4°C for 2 h. The immune complexes were immobilized on protein A/G agarose beads (Pierce) for an additional 2 h. The resin was washed three times with cell lysis buffer and eluted with SDS sample buffer. Samples were then subjected to Western blot analysis.

RESULTS
Assembly of L27 Domain Heterotrimer-To identify a suitable L27 domain complex for crystallization, several constructs from the same or different species were designed and tested for protein expression and purification. After extensive crystal screening, high-quality crystals were obtained only with the construct containing the L27 domain of rat Patj (L27 Patj ), the tandem L27 domains of human Pals1 ((L27N-L27C) Pals1 ), and the L27 domain of mouse Mals2 (L27 Mals2 ). L27 Patj , (L27N-L27C) Pals1 , and L27 Mals2 were fused into a single polypeptide by two 3C protease-cleavable segments. An additional GGG cassette was inserted before the second 3C protease cleavage segment (supplemental Fig. S1, A and C). This technique, which has been used extensively in the determination of L27 domain structures by our laboratory and others, does not alter the global structure of the complexes (17, 19 -21). The purified L27 Patj /(L27N-L27C) Pals1 /L27 Mals2 complex, both as a singlechain fusion and as three separate chains, eluted as a single peak from an analytical gel filtration column with a molecular mass corresponding to that of the heterotrimer (supplemental Fig.  S2, A-C). Analytical ultracentrifugation confirmed that the L27 Patj /(L27N-L27C) Pals1 /L27 Mals2 complex assembled into a heterotrimer with a molecular mass of ϳ30.4 and ϳ32.3 kDa for complexes with and without covalent linkers, respectively (supplemental Fig. S2, D-G). We conclude that the L27 Patj / (L27N-L27C) Pals1 /L27 Mals2 complex forms a heterotrimer containing four L27 domains.
Structure of L27 Domain Heterotrimer-The structure of the L27 domain heterotrimer was determined using single-wavelength anomalous dispersion. The Fourier map calculated from the initial single-wavelength anomalous dispersion phases showed that the four molecules of the L27 Patj /(L27N-L27C) Pals1 /L27 Mals2 complex were present in one asymmetric unit (supplemental Fig. S3). The four molecules were similar in structure, with a root mean square deviation of less than 0.61 Å for the 245 C␣ atoms. Therefore, we discuss only molecule A (colored red in supplemental Fig. S3) in the following section.
Although there are similarities in conformation and buried surface area, when assembled in this heterotrimer complex, L27 Patj /L27N Pals1 and L27C Pals1 /L27 Mals2 do not interact with each other (Fig. 3A), indicating that each L27 heterodimer is formed mutually independently. This result contrasts sharply with previous findings that two L27 heterodimers are asymmetric and cooperative in the L27 DLG1 /(L27N-L27C) MPP7 /L27 Mals3 heterotrimeric complex (21). To verify our findings, we performed a GST pulldown assay. Our results showed that L27 Patj bound Pals1 with similar affinities in the absence or presence of Mals2 (Fig. 3B) and vice versa (Fig. 3C).
Surface electrostatic analysis suggested that the assembly of the L27 Patj /L27N Pals1 and L27C Pals1 /L27 Mals2 heterodimers is mediated primarily by extensive hydrophobic interactions inside the core of the complex (Fig. 2D). In addition to hydrophobic interactions, several hydrogen bonds between charged residues are involved in the assembly of the two L27 heterodimers. Detailed interactions in each L27 heterodimer (L27 Patj /L27N Pals1 and L27C Pals1 /L27 Mals2 ) of the L27 Patj / (L27N-L27C) Pals1 /L27 Mals2 complex are shown in Fig. 3A. The residues participating in the interactions are evolutionarily conserved according to structure-based sequence alignment (supplemental Fig. S5).
L27 domains of the same homologous protein from different species are usually conserved. The amino acid sequence identity of L27 Patj among mouse, rat, and human is very high (supplemental Fig. S5A). Within the structural region of L27 Patj (residues Gln 11 -Ser 56 ), only four residues are divergent over 46 amino acids, and these four residues are not involved in binding to L27N Pals1 . Within the structural region of the tandem L27 domains of Pals1 (residues Leu 122 -Glu 229 ) (supplemental Fig.  S5B), there are nine different residues over 108 amino acids across mouse, rat, and human, and these residues are also not involved in interactions between each cognate pair of L27 domains except for one residue (Val 169 versus Ile 169 , which are similar hydrophobic residues). The amino acid sequences of L27 Mals2 from mouse, rat, and human are completely conserved (supplemental Fig. S5C). Taken together, we conclude that the 4-L27 domain-containing heterotrimer from the same species should have a very similar structure to that determined in this study with rat Patj, human Pals1, and mouse Mals2. Furthermore, several structures of cognate pair L27 domains from mixed species have been determined (18 -20).

DISCUSSION
A bioinformatic survey in mammals reveals that L27 domains from various scaffold proteins are categorized into two subfamilies: type A and type B (supplemental Fig. S7) (17). Not only are all L27 monomers unable to specifically self-associate, they cannot interact with distinct monomers from within the same type and can form heterodimers only with monomers from a different type (7,17,33). Previous studies (17,19) have shown that the symmetric L27 homotetramers (dimer of heterodimers) is a unified assembly mode for cognate pairs of type A/type B L27 domain complexes. Under physiological conditions, the L27 domain often forms a tandem L27 domain-mediated heterotrimer to achieve its biological functions as a cell polarity regulator (10,15,34). A previous study of a 4-L27 domain-containing heterotrimer from a tripartite DLG1/MPP7/Mals3 complex showed that the heterodimer of L27C MPP7 /L27 Mals3 has multiple contacts with L27 DLG1 of the L27 DLG1 /L27N MPP7 heterodimer (21), suggesting that the assembly of the L27C MPP7 /L27 Mals3 heterodimer promotes the recruitment of L27 DLG1 and further facilitates the L27 DLG1 /L27N MPP7 heterodimer formation. In support of this idea, a prior study showed that the association of DLG1 with MPP7 required the prior formation of a complex between MPP7 and Mals3 (34). Thus, these studies revealed that the two cognate pairs of L27 heterodimers assemble into heterotrimers in a tandem L27 domain-mediated cooperative mode. It was reported that MPP3(DLG3) and MPP2(DLG2), two Mals-binding proteins, bind DLG1/synapse-associated protein 97 (SAP97) and require both the L27N and the L27C domains of MPP3 and MPP2, respectively (35), indicating that these heterotrimers may also form through the tandem L27 domainmediated cooperative assembly mode.
The L27N domain-binding partners of MPP4 and Pals2 (MPP6) have not yet been identified, and the mechanism of L27 domain-mediated tripartite complex assembly mechanism must be uncovered to fully elucidate their biological functions. Nevertheless, although the assembly mode of heterotrimeric structure is divergent, the 4-L27 domain-containing heterotrimeric structure may represent a general assembly mode for tandem L27 domain-mediated obligate tripartite complexes. This distinct mechanism of tandem L27 domain-mediated assembly of heterotrimeric structures may, in part, reveal the correct assembly mode of L27 domain scaffold proteins during the organization of specific supramolecular protein complexes. This hypothesis remains to be tested by further functional studies in vivo. Such tandem L27 domainmediated multimeric scaffolds provide various nucleation platforms for the organization of suprasignaling complexes that have been implicated in a wide range of elementary cellular processes, including asymmetric cell division, cell polarity control, and the regulation of cytoskeletal dynamics and signal transduction.