Structural Insights Into the Apkc Regulatory Switch Mechanism of the Human Cell Polarity Protein Lethal Giant Larvae 2

Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2

Lior Almagora,b, Ivan S. Ufimtseva, Aruna Ayera,b, Jingzhi Lia,b, and William I. Weisa,b,1

aDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; and bDepartment of Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305

Edited by Wesley I. Sundquist, University of Utah Medical Center, Salt Lake City, UT, and approved March 5, 2019 (received for review December 28, 2018) Metazoan cell polarity is controlled by a set of highly conserved down-regulation of Lgl occurs in various human cancers (18). Lgl proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity has important roles in all aspects of cell polarity, including in the through phosphorylation-dependent interactions with several development of epithelial apical-basal polarity, asymmetrical cell other proteins as well as the plasma membrane. Phosphorylation division, and cell migration. of Lgl by atypical protein kinase C (aPKC), a component of the The molecular basis of Lgl function in cell polarity is poorly partitioning-defective (Par) complex in epithelial cells, excludes Lgl understood, but it is clear that it depends upon aPKC phosphorylation- from the apical membrane, a crucial step in the establishment of dependent cellular localization. In early stages of Drosophila epithelial cell polarity. We present the crystal structures of human epithelial development, when polarity is being established, Lgl is Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. both cytoplasmic and uniformly localized at the cell cortex. At Lgl2 adopts a double β-propeller structure that is unchanged by later stages, aPKC phosphorylation excludes Lgl from the apical aPKC phosphorylation of an unstructured loop in its second β-pro- domain, where Par-6 and aPKC concentrate. This apical exclu- peller, ruling out models of phosphorylation-dependent conforma- sion does not occur in a nonphosphorylatable mutant Lgl, which tional change. We demonstrate that phosphorylation controls the results in aberrant polarity (19). In fully polarized cells, Lgl is direct binding of purified Lgl2 to negative phospholipids in vitro. We located mostly at the lateral membrane, where it actively excludes also show that a coil–helix transition of this region that is promoted Par-6 from the cell cortex (19). A similar spatial and temporal by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylation- localization of Lgl in relation to the aPKC/Par-6 complex is also dependent, implying a highly effective phosphorylative switch for observed in mammalian epithelial cultures (20). membrane association. In a polarized epithelial cell, Lgl colocalizes with the polarity proteins scribble (Scrib) and discs large (Dlg) at the apical margin Lgl | cell polarity | aPKC of the lateral membrane. Experiments in Drosophila have demonstrated a strong genetic interaction among these three genes, he development of structurally and functionally distinct cell indicating that they act together in a common pathway in the Tsurfaces is essential for the proper function of most animal regulation of cell polarity (21). Lgl and Dlg mutations have been cells. The establishment and maintenance of such cell polarity shown to produce similar effects on fly development (21), and a require a set of so-called polarity proteins, whose core compo- nents are conserved throughout metazoa. In epithelial cells, Significance apicobasal polarity is controlled by the spatial and temporal cross-talk between polarity complexes located on the apical or – Epithelial cells have a spatially polarized organization. For ex- basolateral membranes, as well as the cell cell junctions that ample, one surface of an intestinal epithelial cell, called the delineate these membrane domains (1, 2). A central modulator apical side, faces the lumen of the gut and has a membrane of this process is atypical protein kinase C (aPKC) (3). aPKC composition distinct from those of the basolateral sides. robustly interacts with partitioning-defective 6 (Par-6), and they Several proteins that control the development and mainte- are found together in a subapical epithelial region in complex nance of apical-basolateral polarity have been identified, but with the aPKC substrate Par-3 (bazooka in Drosophila) (4, 5). their molecular mechanisms are poorly understood. Lethal The aPKC/Par-6/Par-3 complex is important for the establish- giant larvae (Lgl) is a basolateral polarity protein that is lost ment of apical-basal polarity, as well as for the maturation of selectively from the apical membrane during development, epithelial junctions in Drosophila and mammals. It also has roles due to its phosphorylation by atypical protein kinase C. Here, – in asymmetric cell division (6 8). In Caenorhabditis elegans zy- we describe the 3D structure of Lgl in both its unmodified and gotes, a homologous PKC3/Par-6/Par-3 complex, which localizes phosphorylated states, and show that phosphorylation of Lgl to the anterior half following the entry of the sperm cell, is es- mediates a structural switch that controls its association with sential for anterior/posterior polarity (6). The aPKC/Par-6 com- the plasma membrane. plex also colocalizes with the crumbs/PALS1/PATJ (crumbs/ stardust/discs-lost in Drosophila) polarity complex located at the Author contributions: L.A. and W.I.W. designed research; L.A., I.S.U., and A.A. performed apical domain. This complex is also crucial for the establishment research; I.S.U., A.A., and J.L. contributed new reagents/analytic tools; L.A., I.S.U., and of epithelial apicobasal polarity, and it is likewise regulated by W.I.W. analyzed data; and L.A. and W.I.W. wrote the paper. aPKC phosphorylation (9). The authors declare no conflict of interest. In addition to its roles in the apical polarity complexes, aPKC This article is a PNAS Direct Submission. is responsible for the phosphorylation of other polarity proteins, Published under the PNAS license. including lethal giant larvae (Lgl) (3). First identified in Dro- Data deposition: The atomic coordinates and structure factors have been deposited in the sophila genetic screens, lgl mutant flies develop cancer-like de- Protein Data Bank, www.wwpdb.org (PDB ID codes 6N8P, 6N8Q, 6N8R, and 6N8S). fects, leading to uncontrolled growth of larval brain neuroblasts 1To whom correspondence should be addressed. Email: [email protected]. and imaginal discs (10–13). Lgl is conserved in eukaryotes and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can be found in two isoforms in mammals (14). Its mutations 1073/pnas.1821514116/-/DCSupplemental. result in polarity defects in mice and other animals (15–17), and Published online May 14, 2019.

10804–10812 | PNAS | May 28, 2019 | vol. 116 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1821514116 Downloaded by guest on September 23, 2021 direct low-affinity interaction between the guanylate kinase do- by multiple serine phosphorylations in the aPKC target site could main of human Dlg4 and a Lgl2 peptide containing phosphory- be the major determinant of membrane targeting and/or protein– lated aPKC target sites has been characterized biochemically and protein interaction. structurally (22). Interactions between Scrib and Lgl have also Here, we report crystal structures of human Lgl2 in both its been demonstrated (23), but strong biochemical evidence for the phosphorylated and unphosphorylated forms. Our structural existence of a ternary Lgl/Dlg/Scrib complex is lacking. Interaction results confirm the double β-propeller core structure for Lgl2. of Lgl, controlled by aPKC phosphorylation, has been reported There are no apparent structural variations between the with additional targets, including nonmuscle myosin II (NMII) unphosphorylated and phosphorylated states of the protein. We (15, 24, 25), syntaxin4 (26, 27), and others (28–32). In addition to demonstrate the preferential interaction of Lgl2 protein with aPKC, cytoplasmic Lgl is phosphorylated by the aurora A and B phosphatidylinositol 4,5-bisphosphate (PIP2)-containing mem- kinases at mitosis in epithelial cells, which promotes its mitotic branes that is abolished by aPKC phosphorylation, as previously relocalization (33–35). demonstrated with a polybasic peptide fragment of this loop (41, Budding yeast express the Lgl homolog Sro7, a 1,033-residue 42). We also show that a coil–helix transformation of this region, protein that is essential for polarized exocytosis in bud growth promoted by PIP2, is phosphorylation-dependent, which implies (36). The Sro7 structure (37) comprises 14 WD40 repeats arranged an effective membrane switch mechanism. These results set a in two seven bladed β-propeller barrels. The distant Lgl/Sro7 se- firm basis for understanding the mechanistic roles of Lgl in quence homology (∼10% identity) suggests them to be structurally cell polarity. and functionally related. Mouse Lgl1 was shown to partially rescue low salt tolerance and temperature sensitivity associated with the Results loss of Sro7 and its homolog, Sro77 (38, 39). The role of Lgl in Structure of Lgl2. To explore the structure of Lgl in its different exocytosis in vertebrates has yet to be solidly established, however. functional forms, we crystallized human Lgl2 both in its The target serine-rich sequence for aPKC phosphorylation unphosphorylated state and in its in vitro aPKCι-phosphorylated resides in a peptide region predicted to connect two adjacent state. The human Lgl2 gene encodes a 1,020-amino acid protein WD40 repeats in Lgl (Fig. 1A). The equivalent sequence is (Fig. 1A). Based on a multiple sequence alignment (MSA) of Lgl missing from Sro7, consistent with the absence of its regulation homologs with the structured protein regions of Sro7 (37), the by aPKC. Phosphorylation of Lgl by aPKC changes its cellular Lgl2 construct used in this study was slightly trimmed at its N and –

localization and its protein protein interaction preferences. A C termini; the deleted regions were also predicted to be un- BIOPHYSICS AND major structural rearrangement promoted by phosphorylation structured by Foldindex (43). Crystals of the unphosphorylated COMPUTATIONAL BIOLOGY has been proposed as a mechanistic explanation for this switch Lgl2(13–978) appeared in two forms that yielded useful dif- (40). On the other hand, the electrostatic alteration of this region fraction data. Crystal form 1 (44) diffracted to 3.2 Å, whereas

Fig. 1. Human Lgl2 protein. (A) Cartoon representation of the human Lgl2 primary structure. N and C β-propeller blades, numbered 1–14, appear in blue and yellow, respectively. The region containing the aPKC-targeted serines (residues 641–680) is marked in black, with the seven serine residues detected by mass spectrometry to be phosphorylated by aPKCι shown in red. Each β-propeller blade is counted from the N terminus of strand A to the C terminus of strand D (including internal loops). The C propeller loop regions discussed in the main text are shown in parentheses. Regions excluded from the crystallized Lgl2(13– 978) construct are shown in gray. The dashed arrow represents the spatial proximity of β14C and β14D, which are both part of blade 14. The drawing is scaled for the relative sequence lengths of each structural domain. (B) Cartoon representation of the Lgl2 structure (top view of the pLgl2 crystal form 2 structure is presented). The structure is rainbow-colored to better illustrate the peptide chain continuity (N and C termini are blue and red, respectively). The β-propeller blades are numbered in black, and the individual A–D strands are noted in blade 1. The 10D and 11A ends of the unstructured 10–11 loop at the top side of Lgl2 are indicated.

Almagor et al. PNAS | May 28, 2019 | vol. 116 | no. 22 | 10805 Downloaded by guest on September 23, 2021 form 2 crystals (45) diffracted anisotropically with a maximum limit first aPKC phosphorylation site at S641 discussed below (Fig. of 2.2 Å. Phosphorylated Lgl2 (pLgl2) also yielded crystals, similar 3C). A second region deviating from the canonical β-propeller to the unphosphorylated Lgl2 form 2 crystals (46), and diffracted to fold is the 9CD loop that includes residues 534–576 (Figs. 1A and 1.9 Å. Another crystal form (form 3) of pLgl2 (47) was also 4). This elongated loop folds back along the lateral side of the obtained, and diffracted to 3.9 Å. second β-propeller toward the top and includes a β-strand (res- Phase determination proved exceptionally challenging for the idues 543–547) that pairs in a parallel orientation with strand D Lgl2 crystals. Traditional molecular replacement methods using of the blade. Following this strand, a sequence-conserved part of Sro7 as a search model consistently failed, which was not sur- this loop (residues 548–564, partly missing in the form 2 unphos- prising, given the 10% sequence identity between these proteins. phorylated structure) sits above the top surface level of the second Experimental phasing was also problematic in all of the Lgl2 β-propeller (Fig. 3). This forms a noticeable protrusion at the top crystal forms. However, we were able to determine the structure surface. Interestingly, this part of the 9CD loop is in close of crystal form 1 using a computational method that is described proximity to the observed termini of the regulatory 10–11 loop in an accompanying paper (48). (Fig. 3 C and D). The refined form 1 Lgl2 structure was used as a search model In the three different crystal forms, Lgl2 maintains the same to solve the higher resolution form 2 crystal structures of both relative angle between the two β-propellers (Fig. 2 and SI Ap- unphosphorylated Lgl2 and pLgl2. The electron density maps pendix, Fig. S1), which indicates that the interface between them from these crystals were used to build models that were very is quite rigid. The interface is large, 2,422 Å2, and features ex- similar to the search model, but the higher resolution of crystal tensive van der Waals and hydrogen bond interactions that are form 2 revealed additional details. These included water mole- mediated principally by peptide connections that meander be- cules and other small molecules present in the crystallization tween the domains, such as the connection between blades buffer, as well as an α-helix at the Lgl2 N terminus (residues 14– 14 and 1 near the N terminus. The elongated connection be- 20) that is absent in the form 1 model (48). This α-helix con- tween strands 8A and 8B (Fig. 1A) makes a major contribution tributes to a crystal packing interface in both of the crystal forms to this interface. Just after emerging from strand 8A, this seg- 2 and 3 that is mediated mostly by hydrogen bonds. The crystal ment folds into a long α-helix (α5) that docks between blades form 1 Lgl2 structure includes 827 of 979 amino acids of the Lgl2 8 and 9 and the bottom side of the 9CD loop (Fig. 3 and SI protein construct; there was no electron density for residues 13– Appendix, Fig. S2). Following this helix, a long loop stretches 17, 471–484, 635–707, and 853–858. Both the unphosphorylated over the bottom side of the first propeller, where it contacts and phosphorylated form 2 structures are missing residues 261– residues from blade 7, blade 1, and the C-terminal tail before 263, 472–485, and 631–708, and the loop residues 554–555 are connecting back to strand 8B (Fig. 3 and SI Appendix, Fig. S2). missing in the unphosphorylated form 2 structure. The C- This ring-shaped structure thus appears to contribute to the terminal sequence beyond S937 is also missing from all of the structural rigidity of the interpropeller interface. In addition, an structures. Other than these slight variations in the unstructured exceptionally long loop between blades 8 and 9 (Fig. 1A, partly regions, the structures are highly similar (Fig. 2 A and B). disordered in the structures) contacts blades 6 and 7 in the first The Lgl2 structure comprises 14 antiparallel β-sheets, which β-propeller. Finally, the C-terminal tail of Lgl2, after exiting fold into a couple of seven-bladed β-propeller structures that, β-strand 14C, goes back to the first propeller and interacts with together, resemble an open clamshell (Figs. 1B and 2). The first the bottom side of blades 1 and 2 (Fig. 3 and SI Appendix,Fig.S2). β-propeller of Lgl2 comprises residues 33–379, and residues 380– In accord with their major role in the interdomain interaction, 923 form the second β-propeller. The last β-strand of the second these three peptide regions contribute 74% of the buried in- propeller (14D) is formed by residues 24–29 near the N terminus terface between domains; without them, the interface would only of the protein, thereby connecting the first and last strands of the be 627 Å2. structure with a short peptide link (Fig. 1B). This topology is also In their roles as protein–protein interaction hubs, the key in- observed in other double-barrel β-propeller proteins, including teractions of the WD40 family reside at their protein surface (51, Sro7 (Fig. 2C), and is assumed to contribute to the structural 52). Alignment of metazoan Lgl sequences reveals a striking dis- stability of the protein fold and the tight interaction between the parity in the level of conservation of residues located close to the two propellers (37, 49, 50). The seven antiparallel β-sheets that top side versus those located at the bottom of the two β-propellers form the blades of each propeller have a simple connectivity with (Fig. 4). The bottom surface is poorly conserved, whereas moving neighboring strands contiguous in sequence, with the first strand toward the top, the conservation becomes stronger. Such a pattern (A) closest to the barrel axis and the last at the periphery. This is found in other WD40 β-propeller proteins and originates, in topology orients the AB and CD loops to the “bottom” of the part, from the signature WD40 sequence located at the C-terminal clamshell, whereas the BC and loops connecting strand D to part of the C and D strands (52). In addition, most of the char- strand A of the next blade are oriented to the “top” (Fig. 1B). acterized WD40 β-propeller interactions involve residues at the Whereas the structural framework, including the relative orien- top surfaces (52). Indeed, the loops on the top surface of Lgl2 tation between the β-propellers, is relatively conserved between show the highest conservation scores, with the most conserved Lgl2 and Sro7, the loops at the top and bottom surfaces of the segments residing on the loops of the second β-propeller, in- β-propellers, important for specific interactions, are unique to cluding the protrusion formed by the unique 9CD loop and the each protein (Fig. 2C). highly conserved 8–9 connection. In addition, loops from blades The second β-propeller contains significantly more of the 5–7 in the first propeller contribute to the highly conserved sur- Lgl2 protein sequence than does the first propeller, and presents face. The unstructured 10–11 loop that contains the conserved some irregular features relative to the first propeller. In partic- phosphoserine region, as well as other highly conserved positions, ular, blades 8–11 contain several long insertions between and sits at one end of this conserved surface (Fig. 4B). We speculate within the blades (Figs. 1 and 3). The largest insertion lies be- that this conserved surface is important for interactions with tween blades 10 and 11 (residues 627–713). This peptide region, binding partners that are regulated by phosphorylation on the which includes stretches of highly conserved residues, including nearby phosphoserine loop. the aPKC-targeted serines, is mostly missing from the structure (residues 631–708 in crystal form 2 and residues 635–707 in The Effect of aPKC Phosphorylation on Lgl2. aPKC targets a region crystal form 1). Both ends of this region face the top side of the on the 10–11 loop that includes several highly conserved serine protein, where the ordered structure of the loop ends 6 amino residues (SI Appendix, Fig. S3). Specific aPKC phosphorylation acids (or 10 amino acids for the crystal form 2) N-terminal to the has been reported for the different orthologs of Lgl at three to

10806 | www.pnas.org/cgi/doi/10.1073/pnas.1821514116 Almagor et al. Downloaded by guest on September 23, 2021 N propeller C propeller top A

bottom B

C BIOPHYSICS AND

Fig. 2. Lgl2 structures. Alignment between the unphosphorylated Lgl2 crystal form 2 structure (A and B; magenta), unphosphorylated Lgl2 crystal form 1 COMPUTATIONAL BIOLOGY (rmsd = 0.72 Å, 97.94% structure overlap) (A, cyan), and pLgl2 crystal form 2 structure (rmsd = 0.30 Å, 99.88% structure overlap) (B, blue). (C) Structural alignment between the unphosphorylated Lgl2 crystal form 1 (cyan) and Sro7 (orange, Protein Data Bank ID code 2OAJ) (rmsd = 2.3 Å, 57.99% structure overlap). Top views are shown on the left side, and side views are shown on the right side.

five of these serine residues (20, 27, 53, 54). We examined the unphosphorylated Lgl2 and pLgl2 structures in crystal form 2 are phosphorylation of purified Lgl2 treated with purified aPKCι by virtually identical (Fig. 2B). Moreover, the structure of phos- mass spectrometry (the same sample that was used for crystal- phorylated Lgl2 solved in an additional crystal form (form 3) is lization), and found seven phosphorylated serine residues on the similar to the others, indicating that there is no influence of 10–11 loop (S641, S645, S649, S653, S660, S663, and S680), crystallization conditions or crystal packing on these results (SI which include the previously reported sites (Fig. 1 and SI Ap- Appendix,Fig.S1). Importantly, the loop between blades 10 and 11, pendix, Figs. S3 and S4). The unphosphorylated Lgl2 control containing the target serine phosphorylation sites, is missing in both sample showed no phosphorylation in the same analysis. Pre- the pLgl2 and unphosphorylated Lgl2 structures. Correspondingly, vious work has suggested that a large structural rearrangement of circular dichroism (CD) spectroscopy analysis of Lgl2(626–680), Lgl occurs upon aPKC phosphorylation (40). However, the a protein fragment covering most of the sequence of this loop,

Fig. 3. Large insertion elements in the Lgl2 C β-propeller. Views from the top (A), bottom (B), and sides (C and D) are shown. The different insertion elements are colored as follows: 8AB, yellow; 8–9, cyan; 9CD, blue; 10–11, green. The C-terminal region is colored orange.

Almagor et al. PNAS | May 28, 2019 | vol. 116 | no. 22 | 10807 Downloaded by guest on September 23, 2021 A B N propeller C propeller

(10-11) loop

bottom top

Fig. 4. Sequence conservation in Lgl2. (A) Side view. (B) Surface representation. The top (Upper) and bottom (Lower) surfaces are shown. Sequence conservation was determined by ConSurf based on MSA of 150 Lgl orthologs.

indicates that this region is a random coil in solution for both its of the PM (41, 42). The in vivo PM localization of Lgl was shown to aPKC-phosphorylated and unphosphorylated forms (Fig. 5 A decrease, or completely vanish, when multiple lysines and arginines and B). of this region were mutated to alanine, or when the inner PM PIP2 Previous work has demonstrated the importance of a polybasic and PIP4 were electrostatically blocked or specifically depleted. region in the 10–11 loop in Lgl for membrane targeting (41, 42). This was also shown for isolated polybasic regions of Lgl, which This region contains multiple highly conserved, positively charged displayed reduced binding to negatively charged membrane vesicles residues located within the aPKC phosphorylation target region of both when their positively charged residues were mutated and when Lgl (SI Appendix,Fig.S3). The direct interaction of Lgl with the these peptides were phosphorylated by aPKC (41, 42). To test plasma membrane (PM) was shown to be independent of an intact whether these properties are also found in the intact protein, we cortical actin network but, instead, depends on the presence of tested purified Lgl2(13–978) for its in vitro binding of membrane lipids with negatively charged head groups (41). A preference was vesicles. Unphosphorylated Lgl2 showed direct binding to mem- demonstrated for PIP2 and, to a lesser extent, phosphatidylinositol branes, with preference given to negatively charged PIP2-containing 4-phosphate (PIP4), both of which are enriched in the inner leaflet vesicles (Fig. 6 A and B). In contrast, pLgl2 bound PIP2-containing

A B

Lgl2(626-680) 10000 -1 pLgl2(626-680) dmol

2 0 15 200 220 240 260 cm 10 wavelength, nm -10000 ] deg [

-20000 C PIP2:Lgl2 D 10000 0 Lgl2(626-680) -1 0.1 40 0.3 pLgl2(626-680) dmol 200 220 240 260 0.5 30 2 0 1.1

cm wavelength, nm 1.8 2.5 -helix 20 -10000 3.3 % ] deg 4.3 10

[ 5.4 6.7 0 -20000 0 10203040 %TFE

Fig. 5. Coil–helix transition of the Lgl2 10–11 loop. (A) SDS/PAGE of the purified control and aPKC phosphorylated Lgl2(626–680). Due to its highly basic nature, Lgl(626–680) migrates abnormally slowly in SDS/PAGE. This is moderated by aPKC phosphorylation. (B) The 10–11 loop is unstructured in solution

regardless of its phosphorylation state. CD spectra of Lgl2(626–680) vs. pLgl2(626–680). deg, degrees. (C)PIP2 binding promotes an α-helical conformation of the 10–11 loop. CD spectroscopy of Lgl2(626–680) with increasing 10% PIP2/90% PC membrane vesicles. (D) aPKC phosphorylation significantly reduces α-helix propensity of the 10–11 loop. The α-helix percentage values were calculated from the mean residue molar ellipticity values of CD signals at 222 nm of Lgl2 (626–680) and pLgl2(626–680) at increasing trifluoroethanol (TFE) contents.

10808 | www.pnas.org/cgi/doi/10.1073/pnas.1821514116 Almagor et al. Downloaded by guest on September 23, 2021 A in the presence of phosphatidylserine (PS)-containing negatively S P charged vesicles (42). Our results similarly show a clear transition from random coil to α-helix with increasing amounts of (1) PC S P added PIP2 vesicles (Fig. 5C). A helical wheel projection of the residues around the polybasic region of Lgl2 reveals that most of (2) 5% PIP2 (3) 15% PS the positively charged amino acids lie on one side of the α-helix (SI Appendix, Fig. S5), which would form an electrostatically (5) 30% PS (4) 10% PIP2 favorable site for binding the negatively charged membrane. We next compared the coil–helix transitions of the phosphorylated (6) 15% PIP2 and unphosphorylated Lgl2(626–680) fragments by addition of trifluoroethanol (Fig. 5D). Our results show that aPKC phosphorylation significantly reduces the α-helix propensity of this 1 2 3 4 5 6 protein region. Thus, phosphorylation both neutralizes the posi- Input: tive charges and disrupts formation of a favorable spatial distri- bution of positively charged residues needed for the interaction with negatively charged membranes. B Discussion Although its important role in cell polarity is well established, the mechanism of Lgl in this process is poorly understood. Without PC Input: any evidence for enzymatic activity, Lgl presumably functions as – 10% PIP a dynamic scaffold protein that participates in various protein 2 protein interactions. These interactions depend on its phosphorylation state and its cellular localization. Our data present C the high-resolution structure of a metazoan Lgl. The structure is S P characterized by a stable core with a highly conserved surface 10% PIP2 (pre that is a likely site for multiple protein–protein interactions Input: BIOPHYSICS AND phosphorylation) regulated by phosphorylation. The peptide that connects Lgl blades 10 and 11 contains COMPUTATIONAL BIOLOGY aPKC ATP 1 2 3 conserved serine phosphorylation sites and positively charged amino acids that, upon mutation, disrupt Lgl cellular localization and lead to loss of polarity. Previous work has suggested that this (1) + - region could act as a flexible hinge between the N- and C- (2) terminal regions of Lgl, which, upon phosphorylation, would be + + made rigid by the interaction of the negative phosphate groups (3) - + with positively charged amino acid side chains. This molecular rearrangement in Lgl was proposed to promote dissociation of Lgl from the actin cytoskeleton (40). The structures of Lgl2 in Fig. 6. Lgl2 binds negatively charged membranes and is released by aPKC both its unphosphorylated and aPKC-phosphorylated states rule phosphorylation. Pelleting of Lgl2 with vesicles containing PC and the in- out major structural changes produced by aPKC phosphoryla- dicated fraction of negatively charged PIP2 or PS is shown. Membrane-bound protein is detected in the pellet (P), while the unbound protein is in the su- tion. Rather, it appears that association with the membrane is pernatant (S). (A) Unphosphorylated Lgl2 binds preferentially to negatively mediated by the α-helical polybasic region of the 10–11 loop

charged vesicles, with a higher preference for PIP2-containing vesicles com- sequence, which is flexibly connected to the double-barrel core pared with PS-containing vesicles with similar charge density (compare lanes of Lgl. Moreover, modeling suggests that interaction of the helix 2 and 3 and lanes 4 and 5). (B) Binding of unphosphorylated or aPKC pre-pLgl2 with the membrane would place the core of the protein away – (13 978) to PC or 90:10 (PC/PIP2)vesicles.(C) Vesicle-bound Lgl2 is released from the membrane (SI Appendix, Fig. S6), making it unlikely from the pellet by aPKC phosphorylation. Lgl2-bound vesicle mixtures were that there would be an influence of the membrane itself on the incubated with assay buffer with or without ATP (1 mM final concentration) and aPKCι kinase domain (1:100 kinase/Lgl2 ratio) before membrane pelleting. rest of the structure. Although a phosphorylation-induced structural switch is not supported by our results, the arrangement of multiple serine vesicles weakly and did not bind 1-oleoyl-2-palmitoyl-sn-glycero-3- phosphorylation sites flanked by multiple positively charged phosphocholine (PC) vesicles at all (Fig. 6B). In addition, phos- residues likely produces a major electrostatic on/off switch. In- phorylation by aPKC releases the already bound Lgl2 from PIP2/PC duced by the charge neutralization effect of phosphorylation, this membrane vesicles (Fig. 6C). These results support a mechanism in switch controls the interaction and dissociation of Lgl from which the phosphorylation-dependent dissociation of Lgl from the negatively charged lipid head groups, such as PIP2, that are apical membrane is due to neutralization of the positively charged enriched at the intracellular surface of the PM. Consistent with residues of this loop by neighboring phosphoserines, thereby ab- this model, deletion of this charged region of the loop results in lating the electrostatic association with the membrane. Thus, it loss of membrane localization, and multiple serine-to-alanine appears that local chemical modification of the 10–11 loop by mutations in the 10–11 loop break the switch and the aPKC- aPKC, rather than extensive structural changes in Lgl, drives its dependent exclusion of Lgl from the apical domain (41). Our membrane association. results with in vitro purified Lgl2 protein support this previously To test the structural consequences of membrane interactions described model for the PM localization of Lgl by direct binding on the 10–11 loop, we measured the effect of adding PIP2-con- to the polar membrane. Similar to Lgl, mira, and numb, other taining vesicles (10% PIP2/90% PC) to Lgl2(626–680) using CD aPKC substrate proteins that are excluded from the apical cortex spectroscopy. A short peptide region harboring the C. elegans Lgl by the Par complex also contain positively charged motifs that phosphorylation sites (residues 656–681, corresponding to hu- mediate their cortical localization. As in Lgl, these motifs prefer- man Lgl2 640–665) was previously shown to fold into an α-helix entially bind to negatively charged phospholipids and have proximal

Almagor et al. PNAS | May 28, 2019 | vol. 116 | no. 22 | 10809 Downloaded by guest on September 23, 2021 aPKC phosphorylation sites (55). This suggests a common scheme Protein was then directly loaded onto Mono-Q column preequilibrated with in the mechanism of apical cortical exclusion by aPKC. the same buffer with 0.5 mM EDTA and eluted with an NaCl gradient. At early stages of development, Lgl is uniformly located at the Protein at 2 mg/mL was then incubated overnight with 15 mM MgCl2,1mM cell cortex. At a certain time point, aPKC phosphorylation is ATP, and PDK1 at a 1:50 (PDK1/aPKC) mass ratio. PDK1 phosphorylated protein was then purified by a second Mono-Q column with 20 mM Tris (pH triggered, which results in turning off binding of Lgl to the apical 8.0), 100 mM NaCl, 10% glycerol, and 1 mM DTT. Protein was eluted by an membrane. Enrichment of PIP2 phospholipids at the apical NaCl gradient, peaking at around 300 mM NaCl. Eluted protein was then membrane is induced by the PIP3 phosphatase PTEN during loaded onto a Superdex 200 SEC column with 20 mM Tris (pH 8.0) and epithelial morphogenesis (56). Thus, the laterally localized Lgl 200 mM NaCl. observed at later stages of development might be due to the For Lgl2(626–680) purification, cells expressing a 6xHis–MBP fusion of Lgl2 binding to downstream membrane-localized targets, such as Dlg (626–680) were suspended in Lgl2 buffer2 [20 mM Tris (pH 8.0), 200 mM or Scrib, rather than the PM. Moreover, pLgl peptides have been NaCl, 5% glycerol, 5 mM 2-mercaptoethanol (BME)], with 1 mM EDTA, shown to bind to Dlg4. Further work is required to verify the protease inhibitors, and DNase as described above. Cells were lysed as de- phosphorylation state of Lgl at the lateral membrane and its scribed above. The lysate was incubated for 30 min with 10 mL of amylose functional consequences. resin and eluted in Lgl2 buffer 2 with 10 mM maltose. The eluted protein was purified by preparative Superdex 75 SEC in 20 mM Hepes (pH 8.0), Methods 200 mM NaCl, 5% glycerol, and 5 mM BME, and incubated with TEV protease and λ-phosphatase overnight, as described above. Protein was then DNA Constructs. The DNA encoding the human Lgl2 residues 13–978, followed loaded onto a Mono S cation exchange column and eluted by an increasing by a tobacco etch virus (TEV) protease site and a protein-A tag, was cloned NaCl gradient. Selected fractions containing the free Lgl2(626–680) were into the pVL-1393 insect cell expression vector (57). The DNA of human then further purified by Superdex peptide SEC in Lgl2 SEC buffer. Since Lgl2 aPKCι kinase domain (248–596) was cloned into the pAcHLT-A (BD Biosci- (626–680) does not have tryptophan residues in its sequence, concentration ences) baculovirus expression vector, with an N-terminal 6xHis tag and a TEV of the purified protein was determined by the BCA assay (Pierce). site preceding the kinase sequence. The DNA-coding Lgl2(626–680) was For comparison of the unphosphorylated Lgl2(626–680) vs. pLgl2(626–680) cloned into the MCS1 of the pCDF-Duet (Novagen) Escherichia coli expres- CD spectroscopy signal, an identical amount of the protein at 170 μM was sion vector, with an N-terminal a 6xHis tag, a maltose-binding protein (MBP), incubated with aPKCι kinase domain (1:100 kinase/Lgl2) for 3–4 h at room and a TEV site sequence preceding the Lgl2 sequence. temperature in Lgl2 SEC buffer containing 10 mM MgCl2 with or without 1 mM ATP. Complete phosphorylation of the protein fragment was de- Protein Expression and Purification. For protein expression in Sf9 cells [Lgl2 tectable by a band migration shift in SDS/PAGE (Fig. 5A). The proteins were (13–978), aPKCι], recombinant baculovirus was obtained using BestBac 2.0 then purified from the kinase using Superdex peptide SEC in 20 mM Tris (pH Linearized Baculovirus DNA (Expression Systems), following standard pro- 8.0) and 150 mM NaCl. cedures. Cells were grown in SF9 media infected by the recombinant baculovirus and kept shaking at 27 °C for 48 h before being harvested by centrifugation. Cell pellets were suspended in their appropriate buffers Protein Crystallization and Data Collection. Lgl2 crystals were grown by vapor – (discussed below) and stored at −80 °C. diffusion at 22 °C. The unphosphorylated Lgl2(13 978) protein was crystallized in two conditions, using protein concentrated to 3–5 mg/mL: Condition For expression in E. coli [Lgl2(626–680)], BL21(DE3) cells transformed with A [19.8% PEG 3350, 0.29 M Na SO , 0.1 M Bis-Tris propane (pH 7.5), 3% the expression vector were grown in LB media at 37 °C until reaching an 2 4 methanol] yielded mostly plates with a P422 lattice that were not suitable OD of 0.5. Isopropyl β-D-1-thiogalactopyranoside (0.5 mM) was then 600 for data collection and, occasionally, square pyramid-shaped crystals (crystal added, and cells were grown for additional 12–16 h at 16 °C before being form 1) in space group P2 2 2 , and condition B [18–21% PEG 2000 MME harvested by centrifugation. 1 1 1 (methyl ether 2000), 80–100 mM SPG (2:7:7 succinic acid/sodium dihydrogen For purification of unphosphorylated Lgl2, thawed cells were suspended in phosphate/glycine at pH 6.0)] yielded crystals (crystal form 2) in space Lgl2 buffer 1 [20 mM Tris (pH 8.5), 300 mM NaCl, 10 mM MgCl , 10% glycerol] 2 group C2. containing 1 mM DTT, 1 mM ATP, 1 mM PMSF, 150 nM aprotinin, 1 μM The pLgl2(13–978) was crystalized in two conditions, using protein con- leupeptin, 1 μM E-64 protease inhibitor, and 5 units of DNase, and lysed centrated to 6–12 mg/mL: condition C [25% PEG 1500, 100 mM SPG (pH 8.0) using an Emulsiflex homogenizer. After a 90-min centrifugation at 185,000 × g, – clarified lysate was incubated for 2–4 h with 10 mL of IgG-Sepharose resin, with crystals similar to the form 2 dephosphorylated Lgl2(13 978) crystals] followed by a wash with 300 mL of Lgl2 buffer 1 with 1 mM DTT, 1 mM ATP, and condition D [1.4 M ammonium sulfate, 100 mM Hepes (pH 7.5), 150 mM and protease inhibitors. The resin was then incubated at 30 °C in 50 mL of NaCl, 1.07% 1,6-hexanediol (crystal form 3, space group P41212)]. Harvested the same buffer for 30 min to maximize the dissociation of endogenous crystals were soaked in cryoprotectant solutions (25% ethylene glycol, 10% Sf9 aPKC from Lgl2. After overnight incubation with Lgl2 buffer 1 with TEV glycerol, and 15% glycerol in crystallization solutions for crystal forms 1, 2, protease, cleaved Lgl2 protein was collected from the column flow-through. and 3, respectively) before being frozen in liquid nitrogen for data collec- The NaCl concentration was then diluted threefold, and the protein was tion. Diffraction data were collected under cryogenic conditions at the loaded onto a Mono-Q column with 20 mM Tris (pH 8.5), 100 mM NaCl, Stanford Synchrotron Radiation Laboratory. Unit cell parameters, data collection, and refinement statistics are shown in SI Appendix, Table S1. 10 mM MgCl2, 10% glycerol, and 1 mM DTT. After a wash with the same buffer, the protein was eluted by an increasing NaCl gradient, where Lgl2 peaked at around 188.7 mM NaCl (13.22 mS/cm). Next, the protein was Structure Solution and Refinement. Diffraction data were integrated by XDS dephosphorylated by overnight incubation with λ-phosphatase (New Eng- (59) and scaled by Aimless (60). Due to the anisotropic diffraction, the unphosphorylated Lgl2 and pLgl2 crystal form 2 data were subjected to the land Biolabs) in the manufacturer’s supplied buffer with 1 mM MnCl2 at room temperature. The dephosphorylated protein was purified using a STARANISO Server (Global Phasing Limited) (staraniso.globalphasing.org/ Superdex 200 size exclusion chromatography (SEC) column in 20 mM Tris (pH cgi-bin/staraniso.cgi) to perform an anisotropic cutoff and to apply an an- 8.0) and 200 mM NaCl (Lgl2 SEC buffer). isotropic correction to the data. For pLgl2 preparation, the purified Lgl2 protein at 2.5 μM was incubated Phases for the crystal form 1 data were obtained using the Sro7 structure with aPKCι kinase domain (1:10 kinase/Lgl2) in Lgl2 SEC buffer containing (Protein Data Bank ID code 2OAJ) as described in an accompanying paper (48).

1 mM ATP and 10 mM MgCl2 for 8 h at room temperature. The protein was The resulting model was further refined using the Phenix-Refine program (61). then diluted twofold with 20 mM Tris (pH 8.0) and purified on a Mono-Q For the solution of the other Lgl2 and pLgl2 data, the refined form 1 column preequilibrated with 20 mM Tris (pH 8.0) and 100 mM NaCl. The Lgl2 model was used as a search model for molecular replacement in Phenix- pLgl2 was eluted by an increasing NaCl gradient peaking at around Phaser (61). Structure refinement was done using Phenix-Refine and Buster 213.5 mM NaCl (19.37 mS/cm). Selected peak fractions were then further (62). Refinement statistics are provided in SI Appendix,TableS1. purified by Superdex 200 SEC in Lgl2 SEC buffer. Phosphorylation of Lgl2 was assayed by Pro-Q Diamond phosphoprotein gel stain (Thermo Fisher Scien- Structure Analysis. Lgl2 interface size and content were analyzed by jcPISA tific) (SI Appendix, Fig. S4) and mass spectrometry (discussed below). (63). For doing this, the peptide chains between L379/A340 and H22/P33 were The aPKCι kinase domain was purified as described (58), with the fol- disconnected, saving a model having each β-propeller as a different chain. lowing modifications. TALON resin was used instead of HiTrap HP. Protein Structure 3D alignments were performed using the CLICK server for topology- eluted from the TALON resin was dialyzed against 20 mM Tris (pH 8.0), independent comparison of bimolecular 3D structures (cospi.iiserpune.ac.in/

100 mM NaCl, 10 mM MgCl2, 10% glycerol, and 1 mM DTT at 4 °C overnight. click).

10810 | www.pnas.org/cgi/doi/10.1073/pnas.1821514116 Almagor et al. Downloaded by guest on September 23, 2021 MSA for homologs of the human Lgl2 gene was done by ConSurf in a 4:1 (protein/vesicles) volume ratio. After 30 min of incubation at room sequence-only mode (64) with default parameters [UniRef90, MAFFT (mul- temperature, the mixture was centrifuged at 4 °C for 40 min at 200,000 × g. tiple alignment using fast Fourier transform); E-value = 0.0001, iterations = For the experiment in Fig. 6C, the Lgl2/vesicles mixture was supplemented 1, maximal %ID between sequences = 95, minimal %ID for homologs = 35, with assay buffer (1:5 buffer toLgl2/vesicles mixture), with or without 6 mM 150 sequences]. ConSurf in full mode (65–67) was then run using the MSA ATP and 270 nM aPKCι kinase domain, and incubated at room temperature files from the previous run. for an additional hour before centrifugation. The supernatant of each sample was then removed, and the pellet was resuspended in the same Structure Modeling. The 10–11 loop missing in the crystal structure was volume of assay buffer + 1% Triton X-100. Samples of the separated phases modeled by the ab initio protocol implemented in Rosetta (68) using pair- were analyzed by SDS/PAGE. wise contact restraints predicted by our in-house–developed code for amino acid contact prediction. The contact map is predicted by a deep convolu- CD Spectroscopy. Lgl2(626–680) or pLgl2(626–680) samples were added with

tional neural network that has a similar architecture and performance as increasing amounts of trifluoroethanol or 10% PIP2 (90% PC) vesicles, mixed published models (69). well, and incubated for >2 min before each reading. To minimize the solvent scattering, buffer conditions were adjusted to 10 mM Tris (pH 8.0) and Mass Spectrometry Analysis. Samples were processed and analyzed on an 75 mM NaCl. The relative concentrations of Lgl2(626–680) and pLgl2(626– Orbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer. The Top3 method 680) between the CD experiments were determined using the Bio-Rad Pro- that cycled between CID (collision induced dissociation), ETD (electron tein Assay. To avoid the effect of the altered charge due to phosphorylation transfer dissociation) and HCD (high-energy collisional dissociation) frag- on the assay, this was done for a sample of each treated with λ-phosphatase mentation modes was used. The data were searched against a Spodoptera (New England Biolabs). frugiperda database, along with the human Lgl2 sequence and common Data were collected using a Jasco-j815 CD spectrometer. CD spectra at 195– contaminants from the CRAPome (contaminant repository for affinity 260 nM were averaged from three to six sequential readings of each sample. purification) using Proteome Discoverer 2.2 with Sequest HT, Percolator, Following the appropriate buffer spectra subtraction, values were converted and ptmRS. to mean residue molar ellipticity as described (70). Helix propensity (%helix) was determined by the spectra readings at 222 nM as described (71). Membrane Pelleting Assay. For vesicle preparation, 18:1–16:0 PC, porcine α α brain L- -PIP2, and porcine brain L- -PS (all from Avanti Polar Lipids, Inc.) ACKNOWLEDGMENTS. Mass spectrometry analysis was performed by Lisa were mixed at the desired molar ratio in a glass tube and then kept under Nader at the Proteomics Unit of the Shared Resources (Fred Hutchinson argon flow to evaporate the solvent chloroform or chloroform/methanol. Cancer Research Center). This work was supported by grants from the The evaporated lipid mixtures were stored overnight in a desiccator under Mathers Foundation and the US NIH (Grant MH58570 to W.I.W.). I.S.U. was vacuum and then resuspended in 20 mM Tris (pH 8.0), 1 mM DTT, and supported by NIH Grant GM122543. A.A. was supported by a postdoctoral 200 mM NaCl to reach a total lipid concentration of 5 μM. The solutions were grant from the American Heart Association, Western Division. Diffraction BIOPHYSICS AND

then subjected to 10 cycles of freezing in liquid nitrogen + 2 min of thawing data were measured at the Stanford Synchrotron Light Source (SSRL), SLAC COMPUTATIONAL BIOLOGY in a water bath. The vesicles were then passed 10 times through an extruder National Accelerator Laboratory, which is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under with a 100-nm pore size. Vesicle solutions were kept for few days at 4 °C. Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Pro- For pelleting assays, purified phosphorylated or unphosphorylated Lgl2 gram is supported by the DOE Office of Biological and Environmental – (13 978) was diluted in assay buffer [20 mM Tris (pH 8.0), 200 mM NaCl, and Research and by the NIH, National Institute of General Medical Sciences 1 mM DTT to 6.3 μM (Fig. 6A); 20 mM Tris (pH 8.0) and 300 mM NaCl to (NIGMS) (including Grant P41GM103393). The contents of this publication 5.4 μM (Fig. 6B); and 20 mM Tris (pH 8.0), 300 mM NaCl, and 1 mM MgCl2 to are solely the responsibility of the authors and do not necessarily represent 5.4 μM (Fig. 6C)]. Diluted Lgl2 was mixed with the vesicle preparations at a the official views of the NIGMS or NIH.

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10812 | www.pnas.org/cgi/doi/10.1073/pnas.1821514116 Almagor et al. Downloaded by guest on September 23, 2021