Structural Basis for the Unique Heterodimeric Assembly Between Cerebral Cavernous Malformation 3 and Germinal Center Kinase III

Structure Short Article

Structural Basis for the Unique Heterodimeric Assembly between Cerebral Cavernous Malformation 3 and Germinal Center Kinase III

Xueyong Xu,1 Xiaoyan Wang,1 Ying Zhang,1 Da-Cheng Wang,1,* and Jingjin Ding1,* 1Institute of Biophysics, Chinese Academy of Sciences, National Laboratory of Biomacromolecules, Beijing 100101, People’s Republic of China *Correspondence: [email protected] (D.-C.W.), [email protected] (J.D.) http://dx.doi.org/10.1016/j.str.2013.04.007

SUMMARY vascular lesion of the CNS in humans that causes headaches, seizures, and strokes in midlife (Labauge et al., 2007). The Defects in cerebral cavernous malformation protein gene encoding the CCM3 protein was initially characterized as CCM3 result in cerebral cavernous malformation an apoptosis-related gene (Wang et al., 1999) and later (CCM), a common vascular lesion of the human confirmed as the third CCM-related gene (Bergametti et al., CNS. CCM3 functions as an adaptor protein that 2005). Sequence analysis indicated that CCM3 lacks any known interacts with various signal proteins. Among these catalytic domains. Biochemical studies further demonstrated partner proteins, germinal center kinase III (GCKIII) that CCM3 functions as a typical adaptor protein. Together with the other CCM-related gene products KRIT1 (Krev1/ proteins have attracted significant interest because Rap1A interaction trapped 1, also known as CCM1) and OSM GCKIII-CCM3 interactions play essential roles in (osmosensing scaffold for MEKK3, also known as CCM2), vascular physiology. Here, we report the crystal CCM3 could organize a large CCM signaling complex (Hilder structures of CCM3 in complex with the C-terminal et al., 2007; Voss et al., 2007). By recruiting germinal center regulatory domains of GCKIII (GCKIIIct) at 2.4 A˚ kinase III (GCKIII) proteins, CCM3 plays a role in the GCKIII resolution. Our results reveal that GCKIIIct adopts a cellular signaling pathway (Fidalgo et al., 2010; Ma et al., 2007; fold closely resembling that of the CCM3 N-terminal Voss et al., 2007). Furthermore, by interacting with striatins, the dimeric domain. GCKIIIct heterodimerizes with regulatory subunits of PP2A, CCM3 bridges GCKIII kinases CCM3 in a manner analogous to CCM3 homodimeri- with the higher order STRIPAK complex (Goudreault et al., zation. The remarkable structural rearrangement of 2009; Kean et al., 2011). Among these complicated adaptor CCM3 induced by GCKIIIct binding and the ensuing functions, the connection between CCM3 and GCKIII proteins is of great interest. GCKIII is a subfamily of sterile 20-like interactions within CCM3 are characterized as the serine/threonine kinases (STKs) that includes MST4, STK24, structural determinants for GCKIIIct-CCM3 heterodi- and STK25 (Pombo et al., 2007). Increasing physiologic evi- merization. Taken together, these findings provide dence has confirmed that CCM3 is a crucial factor mediating a precise structural basis for GCKIIIct-CCM3 hetero- GCKIII-related vascular development as well as endothelial cell dimerization and the functional performance of junction and lumen formation, which are closely related to the GCKIII mediated by CCM3. etiology of CCM disease (Chan et al., 2011; Voss et al., 2009; Zheng et al., 2010). However, the mechanistic basis governing the interaction between CCM3 and GCKIII remains unclear INTRODUCTION due to the lack of a three-dimensional structure of the GCKIII- CCM3 complex. Adaptor proteins play important roles in numerous signal trans- Previous structural studies have revealed that CCM3 adopts duction pathways. They often determine the specificity of a two-domain architecture and homodimerizes due to the home- signaling and also play indispensable roles in the crosstalk of otypic interactions of the N-terminal dimeric domain. The rigid multiple signaling cascades. They can recruit different partner C-terminal domain contains binding sites for the potential proteins, organize large signaling complexes, and bridge recruitment of partner proteins. The flexible linker connecting upstream and downstream signals. The recruitment of partner the two independent domains allows for a certain amount of proteins by adaptor proteins is dependent on the unique pro- mobility in the overall homodimeric structure (Ding et al., 2010; tein-protein and protein-ligand interaction modules of adaptor Li et al., 2010). Most recently, based on sequence alignment proteins. Structural characterization of the interactions between and binding studies, a report suggested that CCM3 may pre- adaptor proteins and their recruited partner proteins aids our un- ferentially heterodimerize with the C-terminal regulatory domain derstanding of the related signal pathways and their physiologic of GCKIII (GCKIIIct) in a manner that is structurally analogous functions in the cell (Flynn, 2001; Hunter, 2000; Pan et al., 2012). to that of CCM3 homodimerization (Ceccarelli et al., 2011). Loss-of-function mutations in the adaptor protein CCM3 could Here, we present the crystal structures of GCKIIIct-CCM3 com- lead to cerebral cavernous malformation (CCM), a common plexes, which provide the precise structural basis for the unique

Table 1. Data Collection and Refinement Statistics GCKIIIct-CCM3 complexes. The crystal structures of STK25ct- CCM3 and MST4ct-CCM3 complexes were determined at STK25ct-CCM3 MST4ct-CCM3 ˚ ˚ 2.43 A in space group P6522 and 2.40 A in space group Data Collection P41212, respectively (Table 1). Each asymmetric unit contains Space group P6522 P41212 one CCM3 monomer and one STK25ct or MST4ct (Figures 1A Cell Dimensions and 1B). In the structures of the GCKIIIct-CCM3 complexes, a, b, c (A˚ ) a = b = 68.56, a = b = 69.10, the CCM3 monomer consists of eight helices (a1–a8) folded c = 229.27 c = 117.57 into a two-domain architecture, which is similar to that observed a, b, g () a = b = 90.00, a = b = g = 90.00 in the CCM3 homodimer. The N-terminal domain (CCM3nt) com- g = 120.00 prises three helices (a1, a2, and a3) and forms a V-like shape Wavelength (A˚ ) 1.5418 1.5418 beside the C-terminal domain. The C-terminal domain (CCM3ct), Resolution (A˚ ) 59.34–2.43 48.85–2.40 composed of helices a5, a6, a7, and a8, folds into a canonical (2.56–2.43) (2.53–2.40) four-helix bundle, in which all four helices are closely antiparallel Unique reflections 12,822 (1,747) 11,764 (1,666) and amphiphilic, with the nonpolar residues extending their side chains into the core of the bundle. A flexible linker of 27 residues Rmerge (%) 10.6 (32.3) 7.6 (35.3) (residues 71–97 including helix a4) connects the two domains Average I/s(I) 21.4 (7.7) 27.3 (7.4) (Figures 1A and 1B). GCKIIIct, which consists of three or four Completeness (%) 98.9 (95.8) 100.0 (100.0) helices (aA, aB, and aC in STK25ct; aI, aII, aIII, and aIV in Redundancy 12.6 (11.9) 12.8 (12.7) MST4), is clasped together with CCM3nt in the same fashion. Refinement Moreover, GCKIIIct does not directly interact with CCM3ct, Resolution (A˚ ) 41.24–2.43 45.12–2.40 which is consistent with the observations in solution by pull- (2.67–2.43) (2.64–2.40) down assays (Ceccarelli et al., 2011). No. reflections 12,731 (2,987) 11,715 (2,844) a Rwork/Rfree (%) 20.92/26.40 22.52/27.35 GCKIIIct Adopts a CCM3nt-like Structure (23.84/33.14) (26.23/37.10) The three-dimensional structures of the N-terminal domains of No. of Atoms all three GCKIII proteins have been reported; as expected, these Protein 2,055 2,007 structures are very similar to those of other well-known kinase Water 116 112 domains based on their high level of sequence conservation (Re- cord et al., 2010). However, the structures of GCKIII C-terminal Sulfate ion 35 – domains remain obscure. The complex structures reported in B-Factors (A˚ 2) this paper provide a look at the structures of GCKIII C-terminal Protein 20.80 35.35 domains. In the structures of GCKIIIct-CCM3 complexes, Water 20.92 32.13 STK25ct and MST4ct fold into an independent V-shape domain. Sulfate ion 43.91 – Helices aA and aB of STK25ct and helices aI, aII, and aIII of Rmsds MST4ct form one side of the V shape, while helix aC of STK25ct Bond lengths (A˚ ) 0.003 0.004 and helix aIV of MST4ct form the other side (Figure 2A). Struc- Bond angles () 0.684 0.748 tural homolog searches using the Dali server (Holm and Sander, 1995) indicate that the structures of STK25ct and MST4ct closely Dihedral angles () 15.018 15.893 resemble that of CCM3nt (Z-score of 7.0 and 1.5 A˚ Ca root- Chirality () 0.052 0.055 mean-square deviation [rmsd] for residues 356–415 of STK25ct ˚ Planarity (A) 0.003 0.003 aligned with residues 20–70 of CCM3nt; Z-score of 6.4 and 1.3 A˚ Values in parentheses are for the highest-resolution shell. Ca rmsd for residues 352–408 of MST4ct aligned with residues a Five percent of reflections are randomly selected for calculating Rfree. 20–70 of CCM3nt). Structural studies have shown that CCM3nt mediates the homodimerization of CCM3 via homotypic interactions among a series of hydrophobic residues (Ding et al., heterodimerization of CCM3-GCKIII. The potential functional 2010; Li et al., 2010). Both structural superimposition and implications of CCM3-GCKIII heterodimeric assembly are also structure-based sequence alignments indicate that these dimer- discussed. ization-related hydrophobic residues are highly conserved in GCKIIIct (Figures 2A and 2B). Together with previous biochem- RESULTS AND DISCUSSION ical observations in solution (Ceccarelli et al., 2011), it may be rationally concluded that GCKIIIct is the structural determinant Overall Structures of GCKIIIct-CCM3 Complexes for GCKIII homodimerization and that GCKIII kinases could It is known that each GCKIII kinase is composed of an N-terminal form homodimers via GCKIIIct homodimerization, which is catalytic domain (GCKIIInt) and a C-terminal regulatory region similar to the means by which CCM3nt forms homodimers. (GCKIIIct) (Pombo et al., 2007). Previous biochemical studies have indicated that CCM3 directly interacts with GCKIIIct GCKIII Kinases Heterodimerize with CCM3 in a Manner (Ceccarelli et al., 2011; Fidalgo et al., 2010). Accordingly, the Analogous to CCM3 Homodimerization C-terminal regulatory regions of two GCKIII proteins, STK25ct In the structures of GCKIIIct-CCM3 complexes, the interactions and MST4ct, were isolated and utilized in structural studies of between GCKIIIct and CCM3nt are highly similar to the

Figure 1. Structural Overview of the GCKIIIct-CCM3 Complexes (A) Overall structure of the STK25ct-CCM3 complex in scheme and surface presentations. STK25ct is orange, while CCM3nt and CCM3ct are green. The long linker connecting CCM3nt and CCM3ct is yellow. Secondary structure elements are labeled. (B) The MST4-CCM3 complex in the same orientation as the complex in panel (A). MST4ct is magenta. CCM3nt and CCM3ct are green.

CCM3 heterodimers with its structure in CCM3 homodimers indicates that both domains of CCM3 are virtually the same in homo- and heterodimers. Interestingly, the position of a4 relative to CCM3ct in homo- and heterodimers is also quite similar, which likely results from the direct hydrophobic interaction of Leu81 of a4 with the a5–a8 hydrophobic cleft of CCM3ct (Figure S2). However, in interactions between the two CCM3nts in a CCM3 homodimer. GCKIIIct-CCM3 heterodimers, CCM3 rotates 120 between Both GCKIIIcts adopt a CCM3nt-like structure, are reverse-ori- CCM3nt and CCM3ct around the axis perpendicular to aAof ented relative to CCM3nt, and complementarily interact with STK25ct or aII of MST4ct (Figure 3C). The switch controlling CCM3nt (Figures S1A and S1B available online). Superimposi- this remarkable structural rearrangement is the a3–a4 loop (res- tion of the GCKIIIct-CCM3 complex on a CCM3nt homodimer idues 71–75) within the long flexible linker region connecting illustrates that GCKIIIct is well superimposed on one of the CCM3nt and CCM3ct (Figure 3D). In the CCM3 homodimer, CCM3nts in homodimer (Figure 3A). In other words, to complex this loop folds into a helix preceding a4 and combines with a4 with CCM3, GCKIIIct takes the place of one CCM3nt in the to form an extended helix. The resulting extended helix is CCM3 homodimer and therefore heterodimerizes with the other antiparallel to the N-terminal region of a1 of the other CCM3 CCM3nt in a manner analogous to CCM3 homodimerization. monomer within the dimer. In this extended helix, the hydropho- The structural observation that GCKIIIct heterodimerizes with bic residues Val72, Phe76, Leu80, and Met83 interact with CCM3nt is consistent with previous biochemical studies indi- Val25, Ala24, Pro21, Met20, Val18, and Met17 in the antiparallel cating that MST4 and CCM3 form heterodimers in solution N-terminal region of a1 via hydrophobic interactions, stabilizing (Ceccarelli et al., 2011). Here, we also confirmed that STK25ct the orientation of this extended helix and the following CCM3ct and CCM3 form heterodimers in solution by analytical ultra- (Figure 3E). However, in the GCKIIIct-CCM3 complexes, the centrifugation (Figure 3B). Mutation of the four hydrophobic N-terminal region of a1 of GCKIII lacks a corresponding hydro- residues (Leu44, Ala47, Ile66, and Leu67) within the CCM3 phobic surface (which would be required to stably interact dimerization interface to Asp (CCM3 LAIL-4D mutant) abolished with a4) due to the low sequence conservation in this region of CCM3 homodimerization as well as GCKIIIct-CCM3 hetero- GCKIIIct (Figure 3E). As a result, the a3–a4 loop in both com- dimerization (Ceccarelli et al., 2011). In our GCKIIIct-CCM3 plexes changes its conformation to an unfolded state, thus complex structures, these residues directly participate in acting as a switch for the remarkable structural rearrangement GCKIIIct-CCM3nt interactions; thus, our results provide struc- of CCM3 (Figure 3C). In fact, the a3–a4 loop is intrinsically flex- tural evidence for the indispensable roles of these hydrophobic ible because it also confers domain mobility in the CCM3 homo- residues in GCKIIIct-CCM3nt heterodimerization. dimer even when fully folded (Ding et al., 2010; Li et al., 2010).

Remarkable Structural Rearrangement of CCM3 Structural Determinants Stabilizing the Preferential Induced by GCKIIIct binding GCKIIIct-CCM3 Heterodimers Biochemical studies have revealed that both CCM3 and GCKIII The structural rearrangement of CCM3 induced by GCKIIIct ﬁt a monomer-dimer equilibrium model in solution and that binding results in the movement of a4 to a different position so CCM3 preferentially heterodimerizes with GCKIII (Ceccarelli that it has close contact with the N-terminal region of a1 of the et al., 2011). The resulting CCM3 and GCKIII monomers and same CCM3 monomer (Figure 4A). However, in the GCKIIIct- their common dimeric features provide the prerequisite for CCM3 complexes, a4 of CCM3 does not assume an antiparallel GCKIIIct-CCM3nt heterodimerization. However, the structural orientation relative to the N-terminal region of a1 because it does mechanism governing the preference of CCM3 for heterodimeri- in the CCM3 homodimer. Instead, it adopts a different orientation zation with GCKIII over homodimerization remains unclear. perpendicular to the N-terminal region of a1, and the three Careful comparison of the structure of CCM3 in GCKIIIct- important hydrophobic residues within a4—Phe76, Leu80, and

Figure 2. The Structures of GCKIIIcts Are Homologous with that of CCM3nt (A) Structural schemes of STK25ct, MST4ct, and CCM3nt along with a superimposition of the three. The hydrophobic residues that mediate the homodimerization of CCM3 and their counterparts in GCKIIIct are shown as stick models. (B) Sequence alignment of CCM3nt and GCKIIIcts. Secondary structures of CCM3nt are schematically represented above the sequences. Strictly conserved residues are highlighted with a red background. Similar residues are boxed and light red. The hydrophobic residues that mediate the homodimerizationof CCM3 are marked with black triangles below the sequences.

Met83—are oriented so that their side chains point into the mined that the association rates of the CCM3 FLM-3D mutant V-shaped hydrophobic cleft formed by the N-terminal region of with both GCKIIIcts were comparable to those of wild-type a1 and a3 of CCM3 (Figure 4A). Moreover, the orientation of CCM3. However, we noted that the disassociation rates of the a4 and the following CCM3ct in the GCKIIIct-CCM3 complexes mutant from both GCKIIIcts were increased more than ten times gives rise to direct interactions between the N-terminal region compared with wild-type CCM3 (Figure 4B; Table 2). As a result, of a1 of CCM3nt and the a5–a8 face of CCM3ct. In the CCM3 the binding afﬁnities of the CCM3 FLM-3D mutant for both homodimer structure, the N-terminal region of a1 of CCM3nt GCKIIIcts were reduced ten times compared with those of the does not directly interact with the a5–a8 face of the other wild-type CCM3. These results are consistent with our preceding CCM3 monomer (Ding et al., 2010; Li et al., 2010). The side chain structural analysis. of Tyr23 within the N-terminal region of a1 is rotated approxi- mately 120 relative to its orientation in the CCM3 homodimer Main Structural Basis for the Functionality of GCKIII and forms two hydrogen bonds with His199 and Asn202 within Proteins Mediated by CCM3 a8. Additionally, the side chain of Met20 within the N-terminal Previous biochemical and pathologic studies have implicated region of a1 is inserted into the hydrophobic cleft formed CCM3 in GCKIII signaling and revealed that the CCM3-GCKIII by Leu80 and Ala84 of a4 and Ile198 of a8(Figure 4A). The struc- pathway could mediate complicated physiologic functions tural rearrangement of CCM3 induced by GCKIIIct binding that are important for blood vessel development as well as generates a series of interactions between a4/CCM3ct and the endothelial cell junction and lumen formation (Chan et al., N-terminal region of a1 of CCM3nt. As essential supplements 2011; Voss et al., 2009; Zheng et al., 2010). STK25 localizes to for GCKIIIct-CCM3nt heterodimerization, these interactions pro- the Golgi apparatus and is activated by autophosphorylation to vide extra stability for GCKIIIct-CCM3 assembly, thus resulting regulate Golgi assembly and cell orientation (Preisinger et al., in the formation of the preferential GCKIIIct-CCM3 heterodimer 2004). The interaction between CCM3 and STK25 stabilizes instead of the homodimer. To conﬁrm that these interactions activated STK25 on the Golgi apparatus and further promotes within CCM3 are induced by GCKIIIct binding, we mutated the Golgi assembly and cell orientation (Fidalgo et al., 2010). Mean- three important hydrophobic residues of a4 (Phe76, Leu80, while, CCM3 regulates the balance between MST4 localization and Met83) to Asp, creating the CCM3 FLM-3D mutant, and in the Golgi and in the cytosol by simultaneous binding to we determined the ability of this mutant to bind GCKIIIct. Using MST4 and the STRIPAK complex. Upon disruption of the inter- surface plasmon resonance (SPR) measurements, we deter- action between MST4 and CCM3, MST4 would subsequently

Figure 3. Heterodimerization of GCKIIIct with CCM3 and the Resulting Structural Rearrangement of CCM3 (A) Structural superimpositions of GCKIIIct-CCM3nt heterodimers with the CCM3nt homodimer as viewed from the front (left) and the side (right). The CCM3nt homodimer is gray. (B) The molar mass profiles of wild-type CCM3, the STK25ct-CCM3 complex, and the CCM3 LAIL-4D mutant as determined by analytical ultracentrifugation. (C) Structural superimpositions of GCKIIIct-CCM3 complexes with the CCM3 homodimer indicate a large rotation between CCM3nt and CCM3ct in the GCKIIIct- CCM3 complexes. The remarkable structural rearrangement of CCM3 is highlighted. (D) Close-up view of the molecular switch governing the structural rearrangement of CCM3. (E) Close-up view of the interactions between a1 and a4 in the CCM3-homodimer, which are absent in the GCKIIIct-CCM3 complexes. Hydrophobic residues that are involved in the interactions are represented as stick models. The corresponding residues in GCKIIIct are also shown. Also see Figures S1 and S2. localize to the Golgi apparatus and be activated by autophos- homodimerization. The remarkable structural rearrangement phorylation, thus regulating Golgi assembly and cell orientation of CCM3 induced by GCKIII binding and the ensuing interac- in an opposing manner to that of STK25 (Kean et al., 2011). tions within CCM3 endows the GCKIII-CCM3 heterodimer with Homodimerization has been characterized as a prerequisite greater stability than the homodimer, which in turn regulates for GCKIII autophosphorylation (Preisinger et al., 2004). Here, the functional performance of GCKIII proteins. our GCKIIIct-CCM3 complex structures reveal that GCKIIIct adopts a CCM3nt-like structure, which is a typical dimeric fold EXPERIMENTAL PROCEDURES and has been confirmed to mediate the homodimerization of CCM3 (Ding et al., 2010; Li et al., 2010). Therefore, it is rational Cloning, Expression, and Purification to propose that the structural basis for the autophosphorylation The MST4ct-CCM3 complex was constructed, expressed, and purified as previously described (Xu et al., 2012). The C-terminal regulatory domain of of GCKIII proteins is the homodimerization of their C-terminal human STK25 (amino acids 355–426) was subcloned into a modified pET- regulatory regions. 32a(+) vector with a thioredoxin tag, a His6 tag, and a PreScission protease When CCM3 is recruited to the GCKIII proteins, it hetero- site at the N terminus to facilitate protein folding and purification. The recom- dimerizes with GCKIII in a manner that is analogous to CCM3 binant plasmid was transformed into Escherichia coli strain BL21 (DE3)

Figure 4. Structural Determinants Stabilizing the GCKIIIct-CCM3 Heterodimerization (A) Structural schemes of the interactions within CCM3 induced by GCKIIIct binding. Residues involved in the interactions are shown as stick models. Hydrogen bonds are shown as black dashed lines. (B) Interactions between CCM3 (wild-type and FLM-3D mutant) and GCKIIIct as measured by SPR experiments. Sensorgrams indicate the titrations at different concentrations.

(Novagen, Darmstadt, Germany). Proteins were expressed for 18 hr at 291 K containing 1 ml of protein solution and 1 ml of reservoir solution equilibrated after induction with 0.25 mM isopropyl-b-D-thiogalactoside. Cell pellets ex- over 80 ml of reservoir solution. STK25ct-CCM3 crystals were obtained with pressing recombinant CCM3 and STK25ct were harvested, mixed together, reservoir buffer containing 0.1 M Bis-Tris pH 5.5, 25% w/v polyethylene glycol and lysed by sonication in lysis buffer (20 mM HEPES pH 7.5, 0.4 M sodium 3350, and 0.2 M ammonium sulfate within 2 days. The crystals were soaked in chloride, 5 mM imidazole, and 5 mM b-mercaptoethanol). After centrifugation, a cryoprotecting solution containing 0.1 M Bis-Tris pH 5.5, 30% w/v polyeth- the soluble proteins were ﬁrst puriﬁed using a Ni-NTA chromatography column ylene glycol 3350, 0.2 M ammonium sulfate, and 10% dimethyl sulfoxide

(Novagen). The thioredoxin and His6 tags of STK25ct were cleaved with (DMSO) before being flash-frozen with liquid nitrogen. MST4ct-CCM3 crystals PreScission protease for 10 hr after dialysis with lysis buffer lacking imidazole. were obtained with reservoir buffer containing 0.1 M Bis-Tris pH 6.0, 25% w/v Subsequently, the protein mixture was purified again using a Ni-NTA chroma- polyethylene glycol 3350, and 0.3 M ammonium acetate within 2 days. The tography column (Novagen). The STK25ct-CCM3 heterodimer was separated crystals obtained were transferred through a solution containing 0.1 M Bis- from the thioredoxin and His6 tags by size-exclusion chromatography using Tris pH 6.0, 30% w/v polyethylene glycol 3350, 0.3 M ammonium acetate, a Hiload 16/60 Superdex 75 column (GE Healthcare, Pittsburgh, PA USA). and 10% dDMSO before being flash-frozen with liquid nitrogen. Diffraction The purified protein was concentrated to 60 mg/ml and stored in a buffer data of these two complexes were collected on a Rigaku FR-E diffraction containing 20 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM DTT at 193 K. system using a Rigaku R-Axis IV++ image plate detector at 40 kV and 40 mA. The data were processed with IMOSFLM and scaled with SCALA from Crystallization, Data Collection, and Structure Determination the CCP4 program suite (Dodson et al., 1997). The structures of the two Crystals of STK25ct-CCM3 and MST4ct-CCM3 complexes (60 mg/ml) were complexes were determined by molecular replacement with Molrep from the grown by the sitting-drop vapor diffusion method at 293 K with 2 ml drops CCP4 program suite, and the structure of the C-terminal domain of CCM3

Received: December 11, 2012 Table 2. Kinetics and Affinity Constants for Wild-Type and Revised: March 20, 2013 Mutant CCM3 Proteins Binding to GCKIIIct Accepted: April 8, 2013 Association Dissociation Binding Published: May 9, 2013 À1 À1 À1 Protein Rate ka (M s ) Rate kd (s ) Affinity Kd (M) 3 3 3 À3 3 À7 wt-STK25 8.86 10 1.45 10 1.64 10 REFERENCES FLM-3D 1.10 3 104 1.52 3 10À2 1.38 3 10À6 mutant-STK25 Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., wt-MST4 4.50 3 103 9.65 3 10À6 2.15 3 10À9 Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for automated crystallographic struc- 3 4 3 À4 3 À8 FLM-3D 5.55 10 1.50 10 2.70 10 ture determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954. mutant-MST4 Bergametti, F., Denier, C., Labauge, P., Arnoult, M., Boetto, S., Clanet, M., Coubes, P., Echenne, B., Ibrahim, R., Irthum, B., et al.; Socie´ te´ Française de Neurochirurgie. (2005). Mutations within the programmed cell death 10 was used as a starting model. A model including the remaining residues was gene cause cerebral cavernous malformations. Am. J. Hum. Genet. 76, 42–51. manually built with Coot (Emsley and Cowtan, 2004), and the two structures Ceccarelli, D.F., Laister, R.C., Mulligan, V.K., Kean, M.J., Goudreault, M., were refined with PHENIX (Adams et al., 2002). The quality of the final model Scott, I.C., Derry, W.B., Chakrabartty, A., Gingras, A.C., and Sicheri, F. was checked using MolProbity (Davis et al., 2007). A sequence alignment (2011). CCM3/PDCD10 heterodimerizes with germinal center kinase III was generated using ClustalW (Chenna et al., 2003), and the sequence align- (GCKIII) proteins using a mechanism analogous to CCM3 homodimerization. ment figure was produced using ESPript (Gouet et al., 1999). All other figures J. Biol. Chem. 286, 25056–25064. were rendered in PyMOL (http://www.pymol.org). Chan, A.C., Drakos, S.G., Ruiz, O.E., Smith, A.C., Gibson, C.C., Ling, J., Passi, S.F., Stratman, A.N., Sacharidou, A., Revelo, M.P., et al. (2011). Mutations in Analytical Ultracentrifugation 2 distinct genetic pathways result in cerebral cavernous malformations in Sedimentation velocity measurements were performed on samples containing mice. J. Clin. Invest. 121, 1871–1881. wild-type CCM3, the CCM3 LAIL-4D mutant, and the STK25ct-CCM3 complex. The samples were loaded at concentrations yielding initial A280 Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G., values of 0.8. Ultracentrifugation was performed at 25C in buffer containing and Thompson, J.D. (2003). Multiple sequence alignment with the Clustal 20 mM HEPES pH 7.5 and 150 mM NaCl using an Optima XL-I analytical series of programs. Nucleic Acids Res. 31, 3497–3500. ultracentrifuge (Beckman Instruments, Palo Alto, CA) with an An50-Ti rotor. Davis, I.W., Leaver-Fay, A., Chen, V.B., Block, J.N., Kapral, G.J., Wang, X., The sample was run for 8 hr at a rotor speed of 60,000 rpm. The data were Murray, L.W., Arendall, W.B., 3rd, Snoeyink, J., Richardson, J.S., and analyzed according to the concentration (M) method using the SEDFIT soft- Richardson, D.C. (2007). MolProbity: all-atom contacts and structure valida- ware package. tion for proteins and nucleic acids. Nucleic Acids Res. 35(Web Server issue), W375–W383. Surface Plasmon Resonance Kinetic Assay Ding, J., Wang, X., Li, D.F., Hu, Y., Zhang, Y., and Wang, D.C. (2010). Crystal The SPR assay was performed at 298 K using a Biacore T100 optical biosensor structure of human programmed cell death 10 complexed with inositol- equipped with a CM5 sensor chip. A running buffer containing 20 mM HEPES (1,3,4,5)-tetrakisphosphate: a novel adaptor protein involved in human pH 7.5, 150 mM NaCl, and 0.005% (v/v) Tween-20 was used for all measure- cerebral cavernous malformation. 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Cell , 113–127. work was funded by the Chinese Ministry of Science and Technology 973 Kean, M.J., Ceccarelli, D.F., Goudreault, M., Sanches, M., Tate, S., Larsen, B., program (grants 2011CB910304 and 2011CB911103), the National Natural Gibson, L.C., Derry, W.B., Scott, I.C., Pelletier, L., et al. (2011). Structure- Science Foundation of China (grant 31100535), and the Chinese Academy of function analysis of core STRIPAK Proteins: a signaling complex implicated Sciences (grant KSCX2-EW-J-3). in Golgi polarization. J. Biol. Chem. 286, 25065–25075.

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