Dual recruitment of two CCM2 molecules to KRIT1 suppresses KLF4 expression

Domain: neuroscience
Published: 2026-02-13
Editorial outcome: minor_revision

Abstract
Regulated expression of Kruppel like factor (KLF) transcription factors is essential for normal maintenance of endothelial cells, but loss of either K-Rev interaction trapped 1 (KRIT1) or cerebral cavernous malformations 2 (CCM2) proteins results in significant over-expression of KLF4 protein, causing the cerebrovascular disorder, cerebral cavernous malformations. Here, combining knockdown and reconstitution in an endothelial cell line, with co-immunoprecipitation, biophysical analysis of purified proteins, and co-crystallography, we find that to restrain KLF4 expression, two CCM2 proteins must cluster on a single KRIT1, with the PTB domain of each CCM2 protein binding either the second or third NPxF motif within KRIT1. This clustering of two PTB domains to a single peptide reveals a previously unobserved mechanism for PTB domain recruitment to partner proteins. Overall, our data support a model where clustering of two CCM2 molecules to one KRIT1 is required for normal regulation of expression of KLF4 transcription factor.

Introduction
The Kruppel like factor 2 and 4 (KLF2 and KLF4) transcription factors are essential for normal endothelial cell function, and disrupted expression can manifest as vascular dysplasias with significant clinical sequelae 1–5. Control of KLF2/4 expression is in part associated with K-rev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2) proteins 6–8. Clinically, the loss of either KRIT1 or CCM2 expression leads to KLF2/4 over-expression, with consequent neurovascular phenotypes that manifest as the life-threatening disorder, Cerebral Cavernous Malformations (CCM) 1,7–16. These effects are recapitulated in murine models of CCM 7,17–22, and KLF4 heterozygosity is protective for loss of KRIT1 7. The molecular basis for how this control of KLF2/4 expression is achieved remains unknown, but interactions of KRIT1 and CCM2 are hypothesized to be important for suppressing MAP kinase (MAPK) signaling through the MEKK3-MEK5-ERK5 pathway, resulting in downregulated KLF2/4 1,7–13,23. Nonetheless, the nature of any regulatory KRIT1-CCM2 interaction required to impinge on KLF2/4 expression has remained an open question.

CCM2 is a multidomain protein that contains a phosphotyrosine-binding domain (PTB) and a Harmonin homology domain (HHD) which coordinate simultaneous CCM2 interaction with KRIT1 and MEKK3, respectively 14,24. CCM2 is thus an adaptor that recruits the MAP kinase kinase kinase, MEKK3, to the membrane associated KRIT1, and therefore acts as a central node connecting KRIT1 to the MAP kinase signaling pathway and consequently to KLF2 and KLF4 6,25–28. The structural basis of the CCM2 HHD domain interaction with the MEKK3 N-terminus and PB1 domain is well defined 14,29. Similarly, CCM2-KRIT1 interactions are generally thought of as well defined, with the CCM2 PTB domain binding the short asparagine-proline-any residue-phenylalanine (NPxF) sequence motifs within KRIT1 (Fig. 1a). Although the stoichiometry has not previously been thoroughly assessed, it seems to be a commonly held assumption that CCM2 and KRIT1 associate in a stoichiometric, 1:1 fashion. This assumption does not correlate perfectly with published data.

Previous studies investigating the binary interactions of CCM2 PTB domain with short peptides corresponding to the KRIT1 NPxF motifs suggest that the third of these three motifs is core to the interaction 24. In contrast, yeast 2-hybrid assays indicate that both the second and third of the NPxF motifs may play roles in CCM2 binding 27,30, and co-immunoprecipitation demonstrates that mutation of both is required to disrupt KRIT1-CCM2 interactions 24. The inconsistency of these data obtained by experiments performed using different techniques potentially indicates a more complex mode of interaction, one that has the potential to influence signaling downstream of CCM proteins.

PTB domains are classified into the larger Pleckstrin Homology/PTB superfamily. The domain was identified in the 1990s, and its binding motif identified to be either NPxpY, NPxY or NPxF, where Y indicates tyrosine and pY indicates phosphotyrosine 31,32. The first structures were determined for IRS-1 and Shc, and demonstrated the mode of binding 33,34. Together with these studies, the identification of multiple NPxF/Y/pY motifs in an array of proteins, including the cytoplasmic tails of integrin and receptor tyrosine kinases, and of KRIT1, suggested that simultaneous engagement of multiple NPxF/Y/pY motifs might be possible by multiple PTB domains. Nonetheless, this has never been conclusively demonstrated at a structural level.

In this study, we reveal that regulation of KLF4 protein expression by the CCM protein complex requires dual recruitment of two CCM2 molecules to a single KRIT1. Using knockdown plus rescue studies in EA.hy926 endothelial-like cell lines, we find that mutations in the CCM2 PTB domain that disrupt KRIT1 binding prevent suppression of KLF4 expression, establishing the importance of CCM2-KRIT1 interactions. Furthermore, deletion of either or both of the second or third CCM2-binding NPxF motifs within KRIT1 results in KLF4 expression similar to KRIT1 knockdown. Using co-immunoprecipitation we show that KRIT1 can bridge two CCM2 molecules, and by assessing co-immunoprecipitation we show that deletion of both the second and third KRIT1 NPxF motifs is required to fully disrupt CCM2 binding. We assess the biophysical properties of the interaction, and by size exclusion chromatography with multi-angle light scattering we find a 2:1 stoichiometry of two CCM2 PTB domains binding a single polypeptide corresponding to the second and third KRIT1 NPxY/F motifs. We then determine the co-crystal structure, which reveals that two CCM2 PTB domains coordinate to a single KRIT1 peptide. This is a previously unobserved mechanism of PTB domain clustering to a single peptide, and is potentially applicable to the interactions of other PTB domain proteins. Together, our data demonstrate that two CCM2 molecules cluster on a single KRIT1 and that this 2:1 stoichiometry is required to achieve normal control of KLF4 expression.

Results
It is now well-established that loss of endothelial KRIT1 or CCM2 leads to increased expression of the important transcription factor KLF4 1,7–13,23. The interaction of KRIT1 and CCM2 proteins, mediated via the CCM2 PTB domain binding to the second or third KRIT1 NPxF motif 24,27,30, is thought to be required for regulation of KLF4 expression, but this has not been directly tested. We therefore used a knockdown/reconstitution system in the immortalized endothelial cell line EA.hy926 to probe the importance of CCM2-KRIT1 interactions.

Lentivirally-delivered shRNA targeting the 3′UTR of CCM2 reduced CCM2 protein expression to virtually undetectable levels, and importantly, this was accompanied by a robust increase in KLF4 expression as assessed by immunoblotting of cell lysates (Fig. 1b, c). As CCM2 knockdown impairs EA.hy926 cell proliferation, restricting our ability to conduct re-expression rescue experiments, we used lentiviral transduction to generate EA.hy926 cells stably expressing knockdown-resistant Myc-tagged CCM2 (CCM2-Myc) and subsequently knocked down endogenous CCM2 in these cells. We found that knockdown-resistant CCM2 prevented KLF4 upregulation in response to CCM2 shRNA (Fig. 1b, c), confirming that this is an on-target effect of the shRNA. To examine whether KRIT1 binding to CCM2 is required, we tested two CCM2 mutants, CCM2(I136A, I139A) and CCM2(C210D), each previously shown to be impaired in binding KRIT1 24. Neither mutant prevented the increased KLF4 expression induced by shRNA targeting endogenous CCM2 (Fig. 1b, c). Pulldown with Myc-tag nanobody beads confirmed that the point mutations disrupted binding to endogenous KRIT1 (Fig. 1d). Thus, CCM2 must be capable of binding KRIT1 to suppress KLF4 expression.

CCM2 binding to either the second or third NPxF motif of KRIT1 has been reported 24,27,30. To assess the importance of the KRIT1 NPxF motifs on KLF4 expression, we generated KRIT1 knockdown EA.hy926 cells using shRNA targeting of KRIT1. shRNA targeting KRIT1 only produced a partial knockdown of endogenous KRIT1 (46 ± 4% KRIT1 remaining) but this was nonetheless sufficient to upregulate KLF4 expression (Fig. 2a, b). This effect was reversed in EA.hy926 cells stably expressing knockdown-resistant KRIT1 (Fig. 2a, b). These results provided a platform for us to investigate the role of the NPxF motifs within KRIT1. We therefore generated KRIT1 mutants that disrupted the CCM2 binding regions comprising NPxF2 (compound I229D mutation and deletion of N231PLF residues (ΔNPxF2)) and NPxF3 (V248A, Y252A, and F253A triple mutation (ΔNPxF3)). Unexpectedly, we found that following knockdown of endogenous KRIT1, KRIT1 lacking either NPxF2 or NPxF3 was unable to reduce KLF4 expression to control levels. Instead, in both cases, the expression of KLF4 resembled the expression of KLF4 in the KRIT1 knockdown cells. Similarly, compound mutation of both NPxF2 and NPxF3 did not downregulate KLF4 expression (Fig. 2a, b). These results could not be explained by the lack of expression of the mutant KRIT1, as these were comparable to the WT control KRIT1 (Fig. 2a). Instead, they suggest the possibility that, to control KLF4 levels, CCM2 may need to bind at both the second and third NPxF motif of KRIT1.

Prior reports 1,7–16 point to loss of CCM proteins leading to transcriptional dysregulation of KLF4, resulting in an increase in KLF4 mRNA. We therefore performed real-time quantitative PCR to test whether the increased KLF4 protein expression observed in KRIT1 knockdown cells, and knockdown cells re-expressing mutant KRIT1 (Fig. 2a, b), reflects changes in KLF4 mRNA levels. As shown in Fig. 2c, KRIT1 knockdown significiantly increased KLF4 message levels and while this was reversed in cells re-expressing knockdown-resistant KRIT1, cells re-expressing KRIT1ΔNPxF2, KRIT1ΔNPxF3 or KRIT1ΔNPxF2+3 retain significantly increased KLF4 mRNA (Fig. 2c). We note that while KRIT1 knockdown cells expressing the KRIT1ΔNPxF3 mutant exhibit significantly increased KLF4 levels they do not reach the level seen in un-rescued cells or cells rescued with KRIT1ΔNPxF2, or KRIT1ΔNPxF2+3, possibly suggesting residual CCM2 binding to KRIT1ΔNPxF3. Nonetheless, both protein and mRNA measurements support the importance of the second and third NPxF motifs of KRIT1 in control of KLF4 levels.

To investigate the potential for one KRIT1 molecule to bind two CCM2 molecules, we performed co-immunoprecipitation experiments from lysates of HEK293T cells transiently expressing tagged CCM2 and KRIT1. To avoid possible complications due to bridging via endogenous KRIT1, we used CRISPR/Cas9 to generate a stable clone of KRIT1-deficient HEK293T cells (Supplementary Fig. 1) and co-expressed FLAG-tagged CCM2 (FLAG-CCM2), myc-tagged CCM2 (CCM2-Myc), and GFP-tagged KRIT1 (GFP-KRIT1) along with ICAP1(S10E, S25E)-mCherry which binds to the first of the three NPxY/F motifs in KRIT1 and is required to stabilize cytoplasmic expression of the GFP-KRIT1 35,36. This system allowed us to assess whether KRIT1 can bridge two CCM2 molecules, and thus potentially indicate if a 2:1 stoichiometry might be employed (Fig. 3a).

These experiments show that immunoprecipitation of FLAG-CCM2 results in co-immunoprecipitation of GFP-KRIT1, and also of CCM2-myc (Fig. 3b–d). Importantly, in the absence of GFP-KRIT1, CCM2-myc does not co-immunoprecipitate with FLAG-CCM2 (Fig. 3b–d), suggesting that KRIT1 may indeed bridge the FLAG- and Myc-tagged CCM2 proteins. To assess if the KRIT1 NPxF motifs play a role in this bridging, we introduced the ΔNPxF2 and ΔNPxF3 mutations into GFP-KRIT1. In keeping with prior results suggesting that intact CCM2 can bind either the second or third KRIT1 NPxY/F motifs 24, neither of the individual ΔNPxF2 or ΔNPxF3 mutations resulted in loss of KRIT1 binding, but their combination (ΔNPxF2+3) very strongly inhibited KRIT1 binding (Fig. 3b, c). However, targeting either individual NPxF motif or both motifs dramatically impeded CCM2-Myc co-immunoprecipitation, with CCM2-Myc binding almost completely lost for the NPxF2 mutant, and significantly reduced for the NPxF3 mutant (Fig. 3b, d). The unexplained residual binding of CCM2-Myc to the NPxF3 mutant may explain its partial effect on KLF4 mRNA levels (Fig. 2c). Together, our findings suggest that two CCM2 molecules may simultaneously bind to both the second and third NPxF motifs of KRIT1, generating a complex with 2:1 stoichiometry.

To further assess the potential 2:1 stoichometry of the CCM2:KRIT1 complex we expressed and purified the PTB domain of CCM2 and synthesized a 29mer peptide that spans both the second and third NPxF motifs of KRIT1 (NPxF2+3; 227TCIYNPLFGSDLQYTNRVDKVVINPYFGL) (Fig. 4a). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) revealed a significant shift in molecular weight for the sample that contains both CCM2 PTB domain (predicted molecular weight ~20kDa) and NPxF2+3 peptide compared to a sample containing CCM2 PTB domain and a peptide synthesized to contain only the NPxF3 motif (NPxF3; 241TNRVDKVVINPYFG) 24 (Fig. 4b). The average mass of the CCM2 PTB complex with NPxF3 is 20.8kDa ± 1.1%, approximating to the expected 20.9kDa 24. In contrast, the average mass across the entire elution of the CCM2 PTB complex with NPxF2+3 is 32.0kDa ± 1.1%. This mass is between the 22.7kDa expected for a single CCM2 PTB domain interacting with a single NPxF2+3 peptide that we likely observe later in the elution, and the 42.0kDa expected for two CCM2 PTB domains in complex with a single NPxF2+3 peptide that we likely observe earlier in the elution (Fig. 4b). We interpret these results to support the formation of a multi-dentate interaction between KRIT1 and CCM2 in a purified setting, consistent with concentrations achievable in gel filtration. These results raised the possibility that a structural approach may reveal the molecular basis for CCM2 recruitment to KRIT1. We therefore conducted protein crystallography to probe the formation of the complex.

We determined the co-crystal structure of CCM2 PTB domain in complex with the 29mer NPxF2+3 peptide. In the 3.0 Å crystal structure we found that a single peptide engages two PTB domains (Fig. 4c, Supplementary Table 1 and Supplementary Fig. 2). This 2:1 complex is mediated by two PTB domain interactions with the NPxF motifs that also uses short sequences prior to each NPxF motif. The interactions of both PTB domains with the peptide are similar (Fig. 4d, e and Supplementary Fig. 3), and for both NPxF motifs, the asparagine residue engages in hydrogen bonding to PTB domain carboxyl oxygens, the proline residue enforces a turn in peptide direction, and the phenylalanine residues stack into a hydrophobic pocket at the N-terminus of PTB domain strand β4. For both PTB interactions with the NPxF peptides, we also observe that a short sequence N-terminal to the NPxF motif stacks against the PTB domain helix αB and completes the PTB β-barrel as an anti-parallel β-strand (Fig. 4c–e and Supplementary Fig. 3). Comparison with the previous structure of CCM2 PTB domain in complex with the NPxF3 motif alone shows an near identical interaction between CCM2 and KRIT1 for these residues (Supplementary Fig. 4). This mode of interaction is broadly the canonical mode of PTB interaction with NPxF/Y/pY motifs and demonstrates that each of the NPxF2 and NPxF3 motifs can directly interact with the PTB domain of CCM2 in isolation.

The structure also reveals the molecular basis for coordinated bidentate interaction of CCM2 with KRIT1. The linker between the NPxF motifs (residues Gly235 to Asp245) forms a short α-helix and makes contact with both PTB domains. This contacts the two CCM2 PTB domains in a parallel orientation and the N-terminal loop (residues 55–62) of the NPxF2-binding CCM2. We observe similar stacking of the CCM2 PTB domain in the lattice of a previous CCM2 PTB domain crystal structure (PDB ID: 4WJ7 24) (Supplementary Fig. 5), but in no other previous structures of PTB domains (by PISA server analysis). We note that recurrence of crystal packing in multiple crystal structures supports biological relevance 37.

The PTB domain is a well studied fold that was recognized in the mid 1990s 33,38,39 and is classified into major groups, defined by Dab, Shc, and IRS-1 31,40. The CCM2 PTB falls in the Dab-like subfamily 24. Some studies have suggested that certain PTB domains might be able to recognize binding partner peptides in differing conformations 41, but we do not observe this for CCM2 interaction with KRIT1 (Supplementary Fig. 3). In contrast, no prior studies have interrogated the structural basis for dual recognition of a single peptide by a PTB protein. There are over 30 peptide-bound NMR and crystal structures of PTB domains 42, yet none are determined in a multi-dentate stoichiometry. We have not identified previous observations of two PTB domains interacting with the same extended peptide. This structure may therefore represent a model for dual engagement of PTB domains to tandem NPxF/Y/pY motif partner proteins.

To assess the potential generalization of this model of PTB domain clustering to a single peptide chain, we assessed human proteins that contain NPxF or NPxY motifs separated by a distance of up to 40 amino acids. We chose this spacing because our crystal structure suggests that the distances separating the NPxF motifs may be important for orientation of the PTB domains and their consequent binding (Fig. 1a). We find that there are 18 proteins in the human genome that may be able to achieve a similar orientation of PTB domain interaction with NPxF/Y/pY residues (Supplementary Fig. 6). As KRIT1 is completely conserved and closely packed to CCM2 in the regions between the canonical PTB-binding regions, it is possible that the linker region between NPxF/Y/pY motifs could be important for these potential tandem PTB binding proteins. We speculate that the clustering of CCM2 to KRIT1 may therefore represent a model of tandem PTB domain clustering to binding partners with multiple NPxF/Y/pY motifs.

Methods
Antibodies and expression constructs
Antibodies were purchased from commercial suppliers—sources and identifiers are provided in Supplementary Table 2. Plasmids used in this study are listed in Supplementary Table 3. To generate specific expression constructs, AttB-flanked PCR amplicons were subjected to Gateway BP recombination with pDONR221 (Thermo Fisher) in order to create ENTRY clones. ENTRY clones or ORFeome clones (ORFeome collaboration 57) were subjected to Gateway LR recombination with Destination vectors (pLENTI-PGK-Blast DEST, a gift from Ben Turk at Yale University, pINDUCER20, a gift from Stephen Elledge (Addgene plasmid # 44012) 58, or pEZY3 a gift from Yu-Zhu Zhang (Addgene plasmid #18672) 59) to generate expression constructs. In order to maintain the expression of CCM2-myc close to endogenous levels in stable cell lines, the PGK promoter of the pLENTI-PGK-Blast-CCM2-myc vectors was crippled by removing the proximal half of the promoter flanked by SpeI sites. Mutations were introduced using Quikchange mutagenesis. All constructs were verified by DNA sequencing.

Lentivirus production
Lentiviruses were produced by co-transfecting HEK293T cells with packaging vectors pCMV-dR8.91 (gift from Dr. Srikala Raghavan, University of Glasgow, U.K.) and pMD2.G (viral protein VSV-G expressed under the CMV promoter; Addgene plasmid #12259, a gift from Didier Trono) together with the pLENTI constructs. Viral supernatant was collected 48h and 72h after transfection and filtered with a 0.45-µm filter.

Cell culture and establishment of stable cell lines
HEK293T cells and EA.hy926 cells were cultured in Complete medium (Dulbecco modified Eagle medium high glucose containing L-Glutamine and supplemented with 9% fetal calf serum, non-essential amino acids and sodium pyruvate, all from Thermo Fisher Scientific). Cell lines were negative for mycoplasma as tested with MycoAlert (Lonza)

To generate KRIT1-null HEK293T cells, the sgRNA oligo CCAGTAGATAGTGACCACT was cloned into the pSpCas9(BB)−2A-GFP sgRNA scaffold (Gift from Feng Zhang; Addgene plasmid #48138) 60 and transiently transfected into HEK293T cells using PEI (Linear Polyethylenimine MW 25,000, Polysciences, Inc.). Forty-eight hours after transfection, GFP positive cells were FACS sorted into 96 well plates using a BD FACSMelody, single cell clones were expanded and validated by SDS-PAGE and immunoblotting.

Stable EA.hy926 cell lines were established by transducing with lentiviral particles in the presence of 8 µg ml−1 polybrene (Sigma) for 18h, followed by selection with Blasticidin 10 µg ml−1 for 6 days. For acute knockdown, EA.hy926 cells were infected with shRNA-containing lentiviral particles in presence of 8 µg ml−1 polybrene for 18h, selected with Puromycin 1 µg ml−1 and harvested 4–5 days after infection.

FLAG pulldown
HEK293T cells were transiently transfected using PEI. Forty eight hours after transfection, cells were harvested using Trypsin/EDTA, quenched with Complete medium, washed with PBS, pelleted, and stored at −80°C until use. Cell pellets were thawed and lysed on ice in buffer X (1mM NaVO4, 50mM NaF, 40mM NaPPi, 50mM NaCl, 150mM Sucrose, 10mM Pipes pH 6.8) containing 0.5% Triton X-100, 0.2% deoxycholic acid, and Complete Ultra Protease Inhibitor cocktail (Roche). Lysates were spun at 21,000g for 10min, supernatants were added to pre-equilibrated anti-FLAG M2 beads (Sigma) and incubated at 4°C for 3h. Beads were then washed with Buffer X containing 0.05% Triton X-100 and bound proteins were fractionated by SDS-PAGE in non-reducing conditions. Proteins were analyzed by immunoblotting and imaged on an Odyssey Infrared Imaging System using Image Studio (Li-COR Biosciences).

Myc pulldown
EA.hy926 cells stably expressing CCM2-Myc were harvested using Trypsin/EDTA, quenched with Complete medium, washed with PBS, pelleted, and stored at −80°C until use. Cell pellets were thawed and lysed on ice in buffer X containing 0.5% Triton X-100, 0.2% deoxycholic acid, and Complete Ultra Protease Inhibitor cocktail (Roche). Lysates were spun at 21,000g for 10min, supernatants were added to pre-equilibrated ChromoTek Myc-Trap® Agarose (Proteintech) and incubated at 4°C for 3h. Beads were then washed with Buffer X containing 0.05% Triton X-100, and bound proteins were fractionated by SDS-PAGE in reducing conditions. Proteins were analyzed by immunoblotting and imaged on an Odyssey Infrared Imaging System using Image Studio (Li-COR Biosciences).

Immunoblotting
Proteins fractionated by SDS-PAGE were transferred onto PVDF membranes (BioRad). PVDF membranes were blocked with Tris Buffer Saline containing 5% non-fat dry milk, incubated with primary and secondary antibodies diluted in Tris Buffer Saline containing 0.1% Tween 20 and 5% Bovine Serum Albumin, and imaged on an Odyssey Infrared Imaging System using Image Studio (Li-COR Biosciences).

Total RNA isolation, cDNA generation, and quantitative real-time PCR (qRT-PCR) analyses
Total RNA was extracted from pellets of 1×106 EAhy.926 cells using the RNeasy Plus mini kit and QIAshredder (Qiagen). cDNA was generated from 1 µg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was then conducted using iQTM SYBR® Green Supermix (Bio-Rad) on a CFX ConnectTM Real-Time System (Bio-Rad). The conditions for qPCR were as follows: Denaturation at 95°C for 3min, 40 cycles at 95°C for 10s and 55°C for 30s. At the end of the amplification procedure, a melting curve was added to check the homogeneity of the amplicons. Samples were tested in triplicates and the expression of KLF4 relative to GAPDH was calculated using the Bio-Rad CFX Manager software. Primer sequences: KLF4 forward 5′-AGAGTTCCCATCTCAAGGCA and reverse 5′-GTCAGTTCATCTGAGCGGG; GAPDH forward 5′-GAAGGTGAAGGTCGGAGA and reverse 5′-TTGAGGTCAATGAAGGGC. Results are plotted as KLF4 Expression from 4 independent experiments.

Protein purification and crystallization
cDNA corresponding to human CCM2 amino acid residues 51-228 (UniProt ID: Q9BSQ5) was subcloned into a modified N-terminally hexahistidine tagged pET32 vector and transformed into E. coli Rosetta (DE3) cells (Novagen). Overnight cultures were inoculated into LB media until A600 = 0.6. Protein expression was induced by the addition of 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), and cells were grown at 18°C overnight prior to harvesting. Cell pellets were resuspended in lysis buffer (500mM NaCl, 20mM imidazole, 20mM Tris, pH 8.0) supplemented with 1mM dithiothreitol (DTT), protease inhibitors, and DNase I. Cell lysis was performed by 3 cycles of freeze/thaw in a dry ice/ethanol bath, followed by sonication. The lysates were centrifuged and the supernatant was loaded onto a HisTrap HP column (GE). The CCM2 PTB domain was eluted from the column by lysis buffer supplemented with 500mM imidazole, and the eluted protein was dialyzed overnight against 250mM NaCl, 20mM Tris, pH 8.0, and 1mM DTT in the presence of TEV protease to cleave the 6x-His tag. As a final step of purification, the cleaved CCM2 PTB domain was eluted from a Superdex S75 size exclusion column in buffer containing 250mM NaCl, 20mM Tris, 1mM DTT, pH 8.0. KRIT1 peptide was synthetically produced at the Tufts University Core Facility as a 29mer peptide with sequence 227TCIYNPLFGSDLQYTNRVDKVVINPYFGL255. For crystallization, the CCM2 PTB domain was filtered and incubated in a 1:1 molar ratio with the solubilized peptide for 2h prior to setting up crystallization trials. Initial crystals were obtained from the JCSG+ Suite (Qiagen) condition G8 at 9.2 mg/ml. These were further optimized in a condition comprised of 0.1M Tris HCl, pH 7, 20% PEG 3350. Crystals were cryo-protected in 20% PEG 400 before immersion in liquid nitrogen.

Structure determination and refinement
Data were collected at APS beamline NE-CAT ID-E. Data were processed in HKL2000 61 to 3.0 Å resolution in space group P21. The structure was determined by molecular replacement using Phaser 62 with Chain A of CCM2 PTB structure (PDB ID: 4WJ7) as the search model. After initial stages of refinement, positive difference density was observed at the NPxF binding site for each of the four PTB chains in the asymmetric unit. The peptide sequences were manually built into this density, and the electron density allowed for unambiguous determination of the register of each peptide. Refinement was conducted using Refmac 63 and Phenix 64, and model building was conducted using Coot 65. The structure was refined to convergence, with final R/Rfree values of 24.7%/29.7%. The final model had good geometry, with a MolProbity score of 1.97 (99th percentile) 66. All crystallographic software was compiled by the SBGrid consortium 67.

SEC-MALS
Purified CCM2 PTB domain at a concentration 51 µM was mixed with the solubilized 29mer KRIT1 NPxF2-3 peptide or NPxF3 peptide in a 1:1 molar ratio. After incubation on ice for 2h, the sample was filtered, and 100 μL was injected onto an SRT-300 column (Sepax Technologies) at 0.4 ml/min in a buffer comprised of 150mM NaCl, 20mM Tris, pH 8.0, 1mM TCEP, and 0.02% Sodium azide. The column was run on an HPLC system (Agilent Technologies 1260) in-line with DAWN-HELEOS II multi-angle light scattering detector (Wyatt Technology) and Optilab T-rEX differential refractometer (Wyatt Technology). Data were analyzed using the ASTRA 6 software from Wyatt Technology.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "How the CCM complex negatively regulates MEKK3-KLF4 signaling remains an open question despite significant advances in the field. Huet-Calderwood and colleagues examine the CCM2-KRIT1 interaction in detail using classical biochemical and structural approaches combined with expression of KLF4, one of the major downstream effectors known to be regulated by the CCM complex. Their studies are clearly described, and definitively show an unusual arrangement in which 2 CCM2 molecules must simultaneously bind one KRIT1 protein via neighboring NPxY/F domains to negatively regulate MEKK3-KLF4 signaling. There is a nice concordance between biochemical and structural data. The conclusions are very solid and the writing is both clear and precise. As the authors note in their discussion, precisely how these findings elucidate CCM complex regulation of MEKK3 is not yet clear, but they are an important clue and will hopefully serve as a foundation for further studies. This reviewer has only minor questions and suggestions that might improve the context of this work.\nClinical/human genetic correlation. Mutations in the KRIT1 NPxY mutations have been associated with CCM disease. The authors’ findings suggest that loss of either CCM2-binding motif would produce an identical loss of function phenotype. Is that supported by human genetic studies and/or mouse genetic studies?\nWhat is the relevance of this finding for regulation of MEKK3 activity by the CCM complext? This key question is rather cursorily discussed in the penultimate paragraph of the discussion, and deserves more thoughtful discussion. The authors note that clustering often activates RTKs, but in that context there is a well-defined activation mechanism so the relevance is not clear. Could the requirement for bi-CCM2 binding reflect a spatial constraint on MEKK3 engagement of substrate? Along these lines, do the authors know of any information that would address whether the spacing of the two adjacent CCM2-binding NPxY motifs is critical for MEKK3 inhibition? E.g., would a KRIT1 peptide with more widely spaced NPxY motifs still bind 2 CCM2 proteins but fail to inhibit MEKK3 activity/KLF4 expression?\nImplications for CCM2L function? CCM2L is a protein homologous to CCM2 that can also bind KRIT1, but the function of CCM2L remains poorly defined. Have the authors attempted heterologous studies to determine how CCM2L binding to KRIT1 affects CCM2 binding and vice-versa? Given the very specific nature of these studies, this would be a natural extension that could yield interesting new insights into how endothelial cells utilize CCM2L to regulate CCM complex function.", "reviewer_2": "The manuscript by Huet-Calderwood et al entitled “Cerebral Cavernous Malformations Protein Complex Suppression of KLF4 Requires Dual Recruitment of Two CCM2 Molecules to KRIT1” focuses on the binding stoichiometry of two scaffolding proteins, KRIT1 and CCM2, and the role this interaction has in regulating the transcription factor KLF4. Through classic biochemical techniques, the authors conclude that two molecules of CCM2 bind to KRIT1, via binding of the CCM2 PTB domain to two adjacent NPXY motifs in KRIT1, and that the binding of 2 CCM2 molecules is required for the ability of this complex to limit expression of KLF4. Overall, the major results of the manuscript support these conclusions, though there are a few observations that do not completely fit this model. The paper is likely to be of interest to researchers studying CCM and this complex, but it is unclear whether it would appeal to a wider audience. The authors propose that the binding modality described herein a may be a model for clustering of PTB domains to proteins containing adjacent NPXY motifs, but this is speculative.\nSpecific concerns:\n1. For Figure 3, the authors indicate that endogenous KRIT1 expression was suppressed to “avoid complications” however, endogenous CCM2 was not suppressed. Would it not cause the same ‘complications”?\n2. In Figure 3, it appears as though FLAG-CCM2 co-IPs a significant amount of GFP. How this could confound the results is not addressed.\n3. Again, in Figure 3, it appears that the KRIT1 deltaNPxF3 mutant is able to co-IP with both FLAG- and Myc- tagged CCM2. In the results this is referred to as “dramatically reduced”, which it is compared to WT, but it is also significantly higher than the deltaNPxF2 mutant and the double mutant. This should be discussed.\n4. The presentation of the SEC data is confusing. The expected MW for CCM2 PTB with NPxF3 is similar to expected. However, it is unclear what binary complex is being referred to at 22.7kD. Why the average mass of the CCM2 PTB NPxF 2 +3 complex is not as expected and how that impacts the validity of the model should be explained in more detail.\n5. The authors should address any uncertainty to their conclusions caused by the fact that their crystal structure only partially represents the intact proteins.\nMinor issues:\n1. It was difficult to track which antibodies were used for the different western blots as it was not specifically spelled out in either the results or the figure legends. Please consider adding them to either location.\n2. In figure 2b, one of the error bars is cut off at the x axis, and this condition only appears to contain an n=4?", "reviewer_3": "The precise regulation of Kruppel-like factor (KLF) transcription factors has been well established as a critical determinant of endothelial cell homeostasis. Loss of KRIT1 or CCM2, or disruption of their interaction, results in the development of cerebral cavernous malformations and is associated with overexpression of KLF2/4. In this study, using knockdown/reconstitution in endothelial cells, co-immunoprecipitation, biophysical assays, and co-crystallography, the authors showed that suppression of KLF4 requires clustering of two CCM2 proteins on a single KRIT1, with each CCM2 PTB domain binding the second or third NPxF motif of KRIT1. They propose that this newly identified PTB clustering mechanism supports a model wherein the dual binding of CCM2 to KRIT1, through its specific structural configuration, is essential for proper regulation of KLF4. While certain shortcomings in the experimental design temper my enthusiasm for the current manuscript, I believe the work has merit. I would be inclined to recommend acceptance provided that the authors satisfactorily address the concerns outlined below.\n1). The central conclusion presented by the authors is that clustering of two CCM2 molecules with a single KRIT1 is essential for the regulation of KLF4 expression. However, the evidence provided relies exclusively on Western blot analyses, which demonstrate changes in KLF4 protein levels. These findings more directly support a role in regulating KLF4 protein stability rather than transcriptional control. This interpretation appears inconsistent with prior reports showing that loss of either CCM1 or CCM2 leads to dysregulation of KLF4 at the transcriptional level. To strengthen their conclusions and avoid potential misinterpretation, it would be helpful if the authors to include RT-PCR data in each panel to directly assess KLF4 transcript levels.\n2). Likewise, the authors appropriately highlight that previous studies have shown MEKK3 activity to be markedly enhanced upon loss of either KRIT1 or CCM2. In contrast, clustering has been recognized as a regulatory mechanism for certain kinases, particularly in maintaining an inactive state—an intriguing concept that stands in contrast to the well-established relationship between the CCM1/CCM2 complex and MEKK3 activity.\nThe present work can be considered, in part, a structure–function study, as the data suggest—though do not yet conclusively demonstrate—that suppression of MEKK3 activity may be mediated by the proposed clustering of two CCM2 molecules with a single KRIT1. Placing greater emphasis on this mechanistic question, which aligns well with the authors’ strengths, would not only sharpen the overall conclusions but also offer a novel perspective on kinase regulation within the CCM pathway. Elucidating this mechanism holds strong potential to make a significant contribution to the field.\n3). Additionally, as the authors already have access to the necessary expression constructs, their hypothesis regarding the functional clustering of two CCM2 molecules with a single KRIT1 could be further strengthened by examining comparative conformational changes across different CCM1/CCM2 complex assemblies. Such analyses might include CCM1/CCM2 in 1:1 and 1:2 ratios, as well as constructs comprising either the N-terminal or C-terminal portions of the CCM2 PTB domain in complex with CCM1, and vice versa. Assessing the conformational differences among these combinations would provide valuable insights into the structural and functional dynamics of the CCM1/CCM2 complex."}, "author_response": "Responses to reviewer queries for:\nHuet-Calderwood, Fisher et al., “Cerebral Cavernous Malformations Protein Complex\nSuppression of KLF4 By Dual Recruitment of Two CCM2 Molecules to KRIT1”\nNature Communications NCOMMS-25-60420\nReviewer #1 (Remarks to the Author):\nHow the CCM complex negatively regulates MEKK3-KLF4 signaling remains an open question\ndespite significant advances in the field. Huet-Calderwood and colleagues examine the CCM2-\nKRIT1 interaction in detail using classical biochemical and structural approaches combined with\nexpression of KLF4, one of the major downstream effectors known to be regulated by the CCM\ncomplex. Their studies are clearly described, and definitively show an unusual arrangement in\nwhich 2 CCM2 molecules must simultaneously bind one KRIT1 protein via neighboring NPxY/F\ndomains to negatively regulate MEKK3-KLF4 signaling. There is a nice concordance between\nbiochemical and structural data. The conclusions are very solid and the writing is both clear and\nprecise. As the authors note in their discussion, precisely how these findings elucidate CCM\ncomplex regulation of MEKK3 is not yet clear, but they are an important clue and will hopefully\nserve as a foundation for further studies. This reviewer has only minor questions and suggestions\nthat might improve the context of this work.\nRESPONSE: We thank the reviewer for their thoughtful and positive assessment of our study. As\ndetailed below we have revised our manuscript to respond to each of the reviewer’s suggestions.\nClinical/human genetic correlation. Mutations in the KRIT1 NPxY mutations have been associated\nwith CCM disease. The authors’ findings suggest that loss of either CCM2-binding motif would\nproduce an identical loss of function phenotype. Is that supported by human genetic studies\nor mouse genetic studies?\nRESPONSE: We agree with the reviewer on the importance of examining the human and mouse\ngenetic data. While we are not aware of any mouse genetic studies targeting point mutations to\nthe KRIT1 NPxY regions, CCM disease-associated missense KRIT1 mutations have been\n1\nreported just downstream of NPxF2 (L238F) (Domingues et al., 2008) or just upstream of NPxF3\n(V244L) (Fisher et al., 2015), consistent with the importance of both motifs in KRIT1. We have\nupdated the manuscript on pages 11 and 12 to state this explicitly.\nPage 11: “….Our study therefore modifies the understanding of the multi-component CCM\ncomplex of proteins, and demonstrates that two CCM2 molecules must coordinate a single KRIT1\nto suppress KLF4 expression. We note that this model is consistent with the identification\nof independent CCM disease-associated missense mutations in KRIT1 proximal to either\nthe second NPxF motif (L238F) 43 or the third NPxF motif (V244L)24.”\nPage 12: “….and likely provide the structural basis for the previously observed preference of\nCCM2 PTB domain for NPxF3 over NPxF224. Our study also provides a molecular\nunderstanding for how mutations in the KRIT1 NPxF2-3 region may impact signaling in\nCCM disease and potentially coronary artery disease 43. We previously showed that the\ndisease-associated V244L mutation in KRIT1 NPxF3 impairs CCM2 binding24, and based\non our current study the 2:1 stoichiometry would be lost for this mutant.”\nDomingues F, Gasparetto EL, Andrade R, Noro F, Eiras A, Gault J, Correia CES, de Souza JM.\nFamilial cerebral cavernous malformations: Rio de Janeiro study and review of the\nrecommendations for management. Arq Neuropsiquiatr. 2008;66(4):795-9.\nFisher OS, Liu W, Zhang R, Stiegler AL, Ghedia S, Weber JL, Boggon TJ. Structural basis for the\ndisruption of the Cerebral Cavernous Malformations 2 (CCM2) interaction with Krev Interaction\nTrapped 1 (KRIT1) by disease-associated mutations. Journal of Biological Chemistry. 2015;\n290(5):2842-53.\nWhat is the relevance of this finding for regulation of MEKK3 activity by the CCM complext? This\nkey question is rather cursorily discussed in the penultimate paragraph of the discussion, and\ndeserves more thoughtful discussion. The authors note that clustering often activates RTKs, but\nin that context there is a well-defined activation mechanism so the relevance is not clear. Could\nthe requirement for bi-CCM2 binding reflect a spatial constraint on MEKK3 engagement of\nsubstrate?\nMEKK3 regulation because we do not understand how this might be occurring at the molecular\nlevel. This comment has prompted us to be a little bolder, and we have discussed some ideas\nabout how the 2:1 stoichiometry may be impacting MEKK3 activity. The penultimate paragraph is\nnow updated as below.\nPage 13: “…. It is unusual for activity of a protein kinase to be suppressed upon clustering;\nreceptor tyrosine kinases are the classic example of clustering causing kinase activation, but\nregulation on clustering has been shown to be important for some kinases, particularly the inactive\nstate of another sub-group of the STE kinases, the type I p21-activated kinases50,51. If the 2:1\nclustering has a direct impact on MEKK3 regulation, one possibility is that the localization\nof dual MEKK3 molecules to the KRIT1-CCM2 complex facilitates a directly inhibited state\nof the kinase. An alternative mechanism for the impact on signaling to KLF might be that\nrecruitment of downstream MEK5 to MEKK3 could be impeded by the 2:1 complex. Future\nstudies will therefore be required to fully understand whether, and if so how, clustering of CCM2\nto KRIT1 can regulate catalytic activity of the MAP kinase kinase kinase, MEKK3….”\nAlong these lines, do the authors know of any information that would address whether the spacing\nof the two adjacent CCM2-binding NPxY motifs is critical for MEKK3 inhibition? E.g., would a\nKRIT1 peptide with more widely spaced NPxY motifs still bind 2 CCM2 proteins but fail to inhibit\nMEKK3 activity/KLF4 expression?\nRESPONSE: When we assess the co-crystal structure we find that the KRIT1 peptide is rather\nclosely packed to both CCM2 PTB domains, including in the region between NPxF2 and NPxF3\n(Reviewer Figure 1A). For the NPxF2-3 region there is also very good sequence conservation,\nincluding complete conservation in the residues between the two canonical PTB-binding motifs\n(e.g. residues 235GSDLQYTNRV) (Reviewer Figure 1B). We believe that the packing and\nconservation are indicative of importance for this region and although the primary interactions are\nmediated by the classical KRIT1 NPxF motifs, mutations or insertions within this region will likely\ndeleteriously impact binding (e.g. V244L). Deconvolution of the impacts of dual binding versus\ndirect effects of the specific orientation of the 2:1 complex upon MEKK3 activity and KLF\nexpression may therefore prove to be non-trivial. To address this query in the text we have\n3\nmodified the text on page 10, and obliquely discussed the possibility of direct regulation by the\ncomplex on page 13.\nPage 10: “….there are 18 proteins in the human genome that may be able to achieve a similar\norientation of PTB domain interaction with NPxF/Y/pY residues (Supplementary Fig. 6). As\nKRIT1 is completely conserved and closely packed to CCM2 in the regions between the\ncanonical PTB-binding regions, it is possible that the linker region between NPxF/Y/pY\nmotifs could be important for these potential tandem PTB binding proteins. We speculate\nthat the clustering of CCM2 to KRIT1 may therefore represent a model of tandem PTB domain\nclustering to binding partners with multiple NPxF/Y/pY motifs.”\nPage 13: “…. It is unusual for activity of a protein kinase to be suppressed upon clustering;\nreceptor tyrosine kinases are the classic example of clustering causing kinase activation, but\nregulation on clustering has been shown to be important for some kinases, particularly the inactive\nstate of another sub-group of the STE kinases, the type I p21-activated kinases50,51. If the 2:1\nclustering has a direct impact on MEKK3 regulation, one possibility is that the localization\nof dual MEKK3 molecules to the KRIT1-CCM2 complex facilitates a directly inhibited state\nof the kinase. ….”\nReviewer Figure 1. Assessment of the KRIT1-CCM2 interaction. A) Space filling\nrepresentation of the crystal structure showing the two CCM2 PTB domains in surface\nrepresentation and the KRIT1 peptide as spheres (same orientation as Fig. 4b). B) Alignment of\nthe KRIT1 sequence for residues 227-255 that are co-crystallized.\nKRIT1, but the function of CCM2L remains poorly defined. Have the authors attempted\nheterologous studies to determine how CCM2L binding to KRIT1 affects CCM2 binding and vice-\nversa? Given the very specific nature of these studies, this would be a natural extension that could\nyield interesting new insights into how endothelial cells utilize CCM2L to regulate CCM complex\nfunction.\nRESPONSE: The reviewer raises an interesting point, and we agree that it will be important to\naddress the role of CCM2L in future projects. We have initiated studies on CCM2L in our\nlaboratories but are not yet ready to report results and prefer to leave the focus of this manuscript\non the question of interactions of CCM2 and the KRIT1 NPxF2 and NPxF3 motifs. Nonetheless,\nas CCM2L has a domain arrangement that allows KRIT1 and MEKK3 binding, the reviewer’s\nquestion highlights the importance of acknowledging the potential for CCM2L interactions to\nmodify the 1:2 KRIT1:CCM2 interaction and the possibility of a 1:1:1 KRIT1:CCM2:CCM2L\ncomplex. We have therefore introduced the question of CCM2L into the discussion on pages 12\nand 13. In light of this comment, we have also softened the title of the manuscript to remove the\nword requires: “Cerebral Cavernous Malformations Protein Complex Suppression of KLF4 By\nDual Recruitment of Two CCM2 Molecules to KRIT1.”\nPages 12 and 13: “Our study reveals significant new information on the importance of clustering\ntwo CCM2 molecules to one KRIT1 in the regulation of KLF4 expression. Nonetheless, important\nquestions that should be addressed in future studies, are raised by this work. The CCM2\nhomolog, CCM2L, which is expressed in endothelial cells, contains a conserved PTB\ndomain and also binds the NPxF motifs of KRIT115,52,53. CCM2L function is poorly\nunderstood and CCM2L mutations have not been linked to human CCM disease but,\ndepending on the models and circumstances tested, CCM2L has been described to both\ncompete and cooperate with CCM215,52-54. Our results revealing the importance of tandem\noccupancy of the KRIT1 NPxF2 and 3 motifs raise the potential that CCM2L binding to one\nor both of these sites may influence the nature and stoichiometry CCM2/KRIT1 complexes\nformed, including the possibility of heterotrimeric complexes53, and may thus modulate\ntheir signaling outputs. Future studies will be needed to address these possibilities. In\naddition, CCM2 and CCM2L are adaptor proteins that recruits the MAP kinase kinase kinase,\nMEKK3, to KRIT114,15,24,54.”\nThe manuscript by Huet-Calderwood et al entitled “Cerebral Cavernous Malformations Protein\nComplex Suppression of KLF4 Requires Dual Recruitment of Two CCM2 Molecules to KRIT1”\nfocuses on the binding stoichiometry of two scaffolding proteins, KRIT1 and CCM2, and the role\nthis interaction has in regulating the transcription factor KLF4. Through classic biochemical\ntechniques, the authors conclude that two molecules of CCM2 bind to KRIT1, via binding of the\nCCM2 PTB domain to two adjacent NPXY motifs in KRIT1, and that the binding of 2 CCM2\nmolecules is required for the ability of this complex to limit expression of KLF4. Overall, the major\nresults of the manuscript support these conclusions, …..\nRESPONSE: We thank the reviewer for their thoughtful comments which we have addressed\npoint-by-point.\n….. though there are a few observations that do not completely fit this model. The paper is likely\nto be of interest to researchers studying CCM and this complex, but it is unclear whether it would\nappeal to a wider audience.\nRESPONSE: We agree that researchers in the CCM field will find this of interest and thank the\nreviewer for this comment. We would also like to highlight the recent broadening of interest in\nthese proteins, and in particular CCM2, by their linkage to coronary artery disease (Schnitzler et\nal., Nature, 2024), and believe that this increases interest in the CCM proteins. From a molecular\nlevel, the ability of PTB domains to cluster to a single peptide has not been observed before, so\nour structural study provides new and novel insights. We have updated the text on page 12 to\nhighlight the recent work of coronary artery disease.\nPage 12: “…likely provide the structural basis for the previously observed differences in CCM2\nPTB domain preferences between NPxF2 and NPxF324. Our study also provides a molecular\nunderstanding for how the KRIT1 NPxF2-3 region may impact signaling in CCM disease\nand potentially coronary artery disease 44. We previously showed that….”\nSchnitzler GR, Kang H, Fang S, Angom RS, Lee-Kim VS, Ma XR, Zhou R, Zeng T, Guo K, Taylor\nMS, Vellarikkal SK, Barry AE, Sias-Garcia O, Bloemendal A, Munson G, Guckelberger P, Nguyen\nMukhopadhyay D, Gupta RM, Engreitz JM. Convergence of coronary artery disease genes onto\nendothelial cell programs. Nature. 2024;626(8000):799-807.\nThe authors propose that the binding modality described herein a may be a model for clustering\nof PTB domains to proteins containing adjacent NPXY motifs, but this is speculative.\nRESPONSE: The observation that two CCM2 PTB domains can directly interact with a single\nKRIT1 molecule is the first time that two PTB domains have been shown to interact with a single\npartner. We have carefully framed our discussion of this observation to indicate that extrapolation\nto other proteins, while speculative, could be of broader interest to the community. To address\nthis concern we have updated text on page 10.\nPage 10: “…. these potential tandem PTB binding proteins. We speculate that the clustering\nof CCM2 to KRIT1 may therefore represent a model of tandem PTB domain clustering to\nbinding partners with multiple NPxF/Y/pY motifs.”\nSpecific concerns:\n1. For Figure 3, the authors indicate that endogenous KRIT1 expression was suppressed to “avoid\ncomplications” however, endogenous CCM2 was not suppressed. Would it not cause the same\n‘complications”?\nRESPONSE: The reviewer is referencing our comment on page 7. In this study, we were testing\nthe ability of KRIT1, or KRIT1 mutants, to bridge two CCM2 proteins. Out of an abundance of\ncaution, to avoid the potential that bridging by endogenous KRIT1 would confound results with\nmutant KRIT1 we generated the KRIT1 deficient cell line. Endogenous CCM2 was not expected\nto bridge, so while it could potentially compete with exogenous CCM2 for binding to KRIT1\nmodestly reducing binding it should not interfere with our ability to detect any bridging via the\nsecond and third NPxF motifs of KRIT1. In response to this comment, we have updated the text\non page 7 to clarify our intent.\nmore detail, we performed co-immunoprecipitation experiments from lysates of HEK293T cells\ntransiently expressing tagged CCM2 and KRIT1. To avoid possible complications due to\nbridging via endogenous KRIT1, we used CRISPR/Cas9 to generate…”\n2. In Figure 3, it appears as though FLAG-CCM2 co-IPs a significant amount of GFP. How this\ncould confound the results is not addressed.\nRESPONSE: As shown in Fig. 3b and Fig. 3c, we observe that FLAG-CCM2 co-IPs significant\namounts of GFP-KRIT1 or the single NPxF mutants and that this is greatly increased over the 2%\ninput shown in the left panel of Fig. 3b. In contrast, only a minimal amount of GFP alone co-IPs\nwith FLAG-CCM2, and this is significantly less than the input GFP. The quantifications shown in\nFig. 3c are normalized for input material and demonstrate that we observe negligible co-IP of\nGFP alone with FLAG-CCM2. We have indicated this quantification in Reviewer Figure 2 below\nand have also made this clearer in the legend for Fig. 3c, and by editing the label for the input\nmaterial in Fig. 3b to read “2% input”. In addition, the negligible binding of the DNPxF2+3 variant\nof GFP-KRIT1 shows that background binding is not a problem in this assay.\nReviewer Figure 2. Reproduction of Fig. 3c with red arrow highlighting quantification of\nco-IPs of GFP alone with FLAG-CCM2.\nLegend for Fig 3c) Quantification of GFP, GFP-KRIT1 or GFP-KRIT1 mutant co-precipitation\nwith FLAG-CCM2 from experiments described in (a). Data were corrected for input levels and\nnormalized to GFP-KRIT1 binding\n8\n3. Again, in Figure 3, it appears that the KRIT1 deltaNPxF3 mutant is able to co-IP with both\nFLAG- and Myc- tagged CCM2. In the results this is referred to as “dramatically reduced”, which\nit is compared to WT, but it is also significantly higher than the deltaNPxF2 mutant and the double\nmutant. This should be discussed.\nRESPONSE: We thank the reviewer for this comment. We agree that while CCM2-Myc binding\nis almost completely lost for the NPxF2 mutant, there is some residual binding for the NPxF3\nmutant. We cannot explain this residual binding but it is consistent and significant (p=0.0373 when\ncompared to the WT protein). In newly performed qPCR assays for KLF4 mRNA levels (Fig. 2c),\nwe also observe that the NPXF3 mutant has a partial effect, possibly due to this unexplained\nresidual binding. We have updated the text on pages 7 and 8 to include a statement about this\n(shown below), have added Fig. 2c (shown below), and have updated Fig. 3d to illustrate this\nsignificance (panel shown below).\nPage 7: “….We note that while KRIT1 knockdown cells expressing the KRIT1DNPxF3\nmutant exhibit significantly increased KLF4 levels they do not reach the level seen in un-\nrescued cells or cells rescued with KRIT1DNPxF2, or KRIT1DNPxF2+3, possibly\nsuggesting residual CCM2 binding to KRIT1DNPxF3. Nonetheless, both protein and mRNA\nmeasurements support the importance of the second and third NPxF motifs of KRIT1 in\ncontrol of KLF4 levels.”\nPage 8: “…However, targeting either individual NPxF motif, or both motifs, dramatically impeded\nCCM2-Myc co-immunoprecipitation, with CCM2-Myc binding almost completely lost for the\nNPxF2 mutant, and significantly reduced for the NPxF3 mutant (Fig. 3b,d). The unexplained\nresidual binding of CCM2-Myc to the NPxF3 mutant may explain its partial effect on KLF4\nmRNA levels (Fig. 2c). Together our findings suggest that two CCM2 molecules may\nsimultaneously bind to both the second and third NPxF motifs of KRIT1; generating a complex\nwith 2:1 stoichiometry.\nstably expressing knockdown-resistant wild-type or mutant KRIT1 cells following KRIT1\nknockdown as in panel a. Bars represent mean +/- SD, n=4. Data were analysed for\ndifferences from the control knockdown condition by one-way ANOVA with Dunnett’s\nmultiple comparisons test. Corrected p values are indicated when significant (p<0.05).\nFig. 3d) Data were corrected for input levels and normalized to results in cells expressing GFP-\nKRIT1. Bars represent mean +/- SD, n=3. Data were analysed for differences from the GFP-\nKRIT1 condition by one-way ANOVA with Dunnett’s multiple comparisons test. Corrected p\nvalues are indicated when significant (p<0.05).\n10\n4. The presentation of the SEC data is confusing. The expected MW for CCM2 PTB with NPxF3\nis similar to expected. However, it is unclear what binary complex is being referred to at 22.7kD.\nRESPONSE: We thank the reviewer for highlighting the imprecise language we used in the\nprevious version of our manuscript. The binary complex at 22.7kDa refers to one made between\na single CCM2 PTB domain and a single peptide encompassing NPxF2+3 motif. We have\nupdated the text on page 8 to make this clearer.\nPage 8: “This mass is between the 22.7 kDa expected for a binary complex of a single CCM2\nPTB domain interacting with a single NPxF2+3 peptide that we likely observe later in the\nelution, ….”\nWhy the average mass of the CCM2 PTB NPxF 2 +3 complex is not as expected and how that\nimpacts the validity of the model should be explained in more detail.\nRESPONSE: We thank the reviewer for suggesting we discuss this in more detail. We note that\nwe have calculated the average mass of the CCM2 PTB-NPxF2+3 complex over the whole elution\npeak. In this calculation across the whole elution peak we believe we have captured a range of\ncomplexes, including those that are approximately the expected molecular weight for a 2:1\ncomplex (red arrow in Reviewer Figure 3), and those that approximately correspond to a 1:1\ncomplex (green arrow in Reviewer Figure 3). For this experiment we do not believe it to be\nappropriate to ‘slice-and-dice’ the elution to fit our expectations for molecular weight, which is why\nwe have presented the data averaged across the peak. To make our interpretation clearer we\nhave updated the text on page 8.\nPage 8: “.…In contrast, the average mass across the entire elution of the CCM2 PTB complex\nwith NPxF2+3 is 32.0 kDa ± 1.1%. This mass is between the 22.7 kDa expected for a single\nCCM2 PTB domain and a single NPxF2+3 peptide that we likely observe later in the elution,\nand the 42.0 kDa expected for two CCM2 PTB domains in complex with a single NPxF2+3 peptide\nthat we likely observe earlier in the elution (Fig. 4b). ….”\napproximately the expected molecular weight for a 2:1 complex and green arrow indicating\nregion with approximately the expected molecular weight for a 1:1 complex.\n5. The authors should address any uncertainty to their conclusions caused by the fact that their\ncrystal structure only partially represents the intact proteins.\nRESPONSE: We thank the reviewer for this comment. We have indeed co-crystallized a portion\nof KRIT1 with a portion of CCM2 and agree with the reviewer that regions outside of these\ncrystallized portions might potentially be of importance. We have therefore modified the text on\npage 12.\nPage 12: “….interactions of both CCM2 PTB domains with KRIT1 are broadly similar to classical\nPTB engagement with NPxF/Y/pY motifs. Although we have co-crystallized portions of KRIT1\nand CCM2, a comparison of the two engagements reveals minor differences in hydrogen-\nbonding between the protein and peptide…..”\nMinor issues:\n1. It was difficult to track which antibodies were used for the different western blots as it was not\nspecifically spelled out in either the results or the figure legends. Please consider adding them to\neither location.\nin Supplementary Table 2 but to facilitate identification of the antibodies used in each figure we\nnow include this information at the end of the legends for Figs. 1, 2 and 3.\nFig. 1. “The following antibodies were used in Fig. 1: anti-KLF4 (Cell Signaling\nTechnologies), anti-CCM2 (Protein Tech), anti-TLK2 (Santa Cruz Biotech), anti-KRIT1\n(AbCam) and anti-Myc (GenScript) (See Supplementary Table 2).”\nFig. 2. “The following antibodies were used in Fig. 2: anti-KLF4 (Cell Signaling\nTechnologies), anti-KRIT1 (AbCam) and anti-TLK2 (Santa Cruz Biotech) (See\nSupplementary Table 2).”\nFig. 3. “The following antibodies were used in Fig. 3: anti-Myc (GenScript), anti-FLAG\n(GenScript) and anti-GFP (Rockland) (See Supplementary Table 2).”\n2. In figure 2b, one of the error bars is cut off at the x axis, and this condition only appears to\ncontain an n=4?\nRESPONSE: We thank the reviewer for noticing this error. The control condition for KRIT1\nexpression null was indeed an N of 5. We have corrected Fig. 2b to show the missing condition\nas per below. In keeping with the journal style, we have also revised our plots to show p values\nfor bars that are significantly different from the CTRL shRNA condition.\nFig. 2b) KLF4 expression levels in experiment described in panel a was assessed. Data are\nnormalized to levels seen in KRIT1 knockdown cells without KRIT1 expression. Bars represent\n13\nmean +/- SD, n=5. Data were analysed for differences from the control knockdown condition by\none-way ANOVA with Dunnett’s multiple comparisons test. Corrected p values are indicated\nwhen significant (p<0.05).\nThe precise regulation of Kruppel-like factor (KLF) transcription factors has been well established\nas a critical determinant of endothelial cell homeostasis. Loss of KRIT1 or CCM2, or disruption of\ntheir interaction, results in the development of cerebral cavernous malformations and is\nassociated with overexpression of KLF2/4. In this study, using knockdown/reconstitution in\nendothelial cells, co-immunoprecipitation, biophysical assays, and co-crystallography, the authors\nshowed that suppression of KLF4 requires clustering of two CCM2 proteins on a single KRIT1,\nwith each CCM2 PTB domain binding the second or third NPxF motif of KRIT1. They propose\nthat this newly identified PTB clustering mechanism supports a model wherein the dual binding\nof CCM2 to KRIT1, through its specific structural configuration, is essential for proper regulation\nof KLF4. While certain shortcomings in the experimental design temper my enthusiasm for the\ncurrent manuscript, I believe the work has merit. I would be inclined to recommend acceptance\nprovided that the authors satisfactorily address the concerns outlined below.\nRESPONSE: We thank the reviewer for their thoughtful and considered comments, which we\nhave addressed below.\n1). The central conclusion presented by the authors is that clustering of two CCM2 molecules with\na single KRIT1 is essential for the regulation of KLF4 expression. However, the evidence provided\nrelies exclusively on Western blot analyses, which demonstrate changes in KLF4 protein levels.\nThese findings more directly support a role in regulating KLF4 protein stability rather than\ntranscriptional control. This interpretation appears inconsistent with prior reports showing that loss\nof either CCM1 or CCM2 leads to dysregulation of KLF4 at the transcriptional level. To strengthen\ntheir conclusions and avoid potential misinterpretation, it would be helpful if the authors to include\nRT-PCR data in each panel to directly assess KLF4 transcript levels.\nRESPONSE: As the reviewer points out numerous prior reports point to loss of CCM proteins\nleading to transcriptional dysregulation of KLF4 – specifically an increase in KLF4 mRNA. This\nincrease in KLF4 message in turn leads to an increase in KLF4 protein, resulting in its functional\neffect on cells. Our measurement of increased KLF4 protein is entirely consistent with this model,\nwhere mRNA levels reflect protein expression levels. However, as the reviewer points out,\nmeasuring protein levels leaves open the alternative possibility of CCM proteins impacting KLF4\n15\nprotein stability. As suggested, we have therefore performed additional RT-PCR to assess KLF4\nmRNA levels in knockdown and rescued cells. These data confirm increased KLF4 transcription\nin KRIT1 knockdown cells and show a substantial rescue of this effect following re-expression of\nwild-type, but not NPxF mutant KRIT1. These data are now shown in a new Fig 2c and discussed\non page 7\nPage 7: “Prior reports1,7-16 point to loss of CCM proteins leading to transcriptional\ndysregulation of KLF4 resulting in an increase in KLF4 mRNA. We therefore performed\nreal-time quantitative PCR to test whether the increased KLF4 protein expression observed\nin KRIT1 knockdown cells, and knockdown cells re-expressing mutant KRIT1 (Fig. 2a,b),\nreflect changes in KLF4 mRNA levels. As shown in (Fig. 2c), KRIT1 knockdown\nsignificiantly increased KLF4 message levels and while this was reversed in cells re-\nexpressing knockdown-resistant KRIT1, cells re-expressing KRIT1DNPxF2, KRIT1DNPxF3\nor KRIT1DNPxF2+3 retained significantly increased KLF4 mRNA (Fig. 2c). We note that\nwhile KRIT1 knockdown cells expressing the KRIT1DNPxF3 mutant exhibit significantly\nincreased KLF4 levels they do not reach the level seen in un-rescued cells or cells rescued\nwith KRIT1DNPxF2, or KRIT1DNPxF2+3, possibly suggesting residual CCM2 binding to\nKRIT1DNPxF3. Nonetheless, both protein and mRNA measurements support the\nimportance of the second and third NPxF motifs of KRIT1 in control of KLF4 levels.”\nFig. 2c) KLF4 mRNA levels were assessed by quantitative real-time PCR in EA.hy926 cells\nstably expressing knockdown-resistant wild-type or mutant KRIT1 cells following KRIT1\nknockdown as in panel a. Bars represent mean +/- SD, n=4. Data were analysed for\ndifferences from the control knockdown condition by one-way ANOVA with Dunnett’s\nmultiple comparisons test. Corrected p values are indicated when significant (p<0.05).\n16\n2). Likewise, the authors appropriately highlight that previous studies have shown MEKK3 activity\nto be markedly enhanced upon loss of either KRIT1 or CCM2. In contrast, clustering has been\nrecognized as a regulatory mechanism for certain kinases, particularly in maintaining an inactive\nstate—an intriguing concept that stands in contrast to the well-established relationship between\nthe CCM1/CCM2 complex and MEKK3 activity.\nThe present work can be considered, in part, a structure–function study, as the data suggest—\nthough do not yet conclusively demonstrate—that suppression of MEKK3 activity may be\nmediated by the proposed clustering of two CCM2 molecules with a single KRIT1. Placing greater\nemphasis on this mechanistic question, which aligns well with the authors’ strengths, would not\nonly sharpen the overall conclusions but also offer a novel perspective on kinase regulation within\nthe CCM pathway. Elucidating this mechanism holds strong potential to make a significant\ncontribution to the field.\nRESPONSE: We agree with the reviewer that this is an intriguing topic, and we have updated our\ndiscussion on page 13 to further discuss some of the potential mechanisms that may be at play.\nWe agree that elucidation of the mechanism of regulation will be of interest to the CCM2 field and\nto multiple others, however, this is a major undertaking that will warrant its own dedicated\npublication at a future date.\nPage 13. “….Our study infers, but does not conclusively demonstrate, that suppression of MEKK3\nactivity is associated with clustering of two CCM2-bound MEKK3 proteins to a single KRIT1. It is\nunusual for activity of a protein kinase to be suppressed upon clustering; receptor tyrosine kinases\nare the classic example of clustering causing kinase activation, but regulation on clustering has\nbeen shown to be important for some kinases, particularly the inactive state of another sub-\ngroup of the STE kinases, the type I p21-activated kinases51,52. If the 2:1 clustering has a\ndirect impact on MEKK3 regulation, one possibility is that the localization of dual MEKK3\nmolecules to the KRIT1-CCM2 complex facilitates a directly inhibited state of the kinase.\nAn alternative mechanism for the impact on signaling to KLF might be that recruitment of\ndownstream MEK5 to MEKK3 could be impeded by the 2:1 complex. Future studies will\ntherefore be required to fully understand whether, and if so how, clustering of CCM2 to KRIT1\ncan regulate catalytic activity of the MAP kinase kinase kinase, MEKK3. ….”\n17\n3). Additionally, as the authors already have access to the necessary expression constructs, their\nhypothesis regarding the functional clustering of two CCM2 molecules with a single KRIT1 could\nbe further strengthened by examining comparative conformational changes across different\nCCM1/CCM2 complex assemblies. Such analyses might include CCM1/CCM2 in 1:1 and 1:2\nratios, as well as constructs comprising either the N-terminal or C-terminal portions of the CCM2\nPTB domain in complex with CCM1, and vice versa. Assessing the conformational differences\namong these combinations would provide valuable insights into the structural and functional\ndynamics of the CCM1/CCM2 complex.\nRESPONSE: The reviewer highlights another intriguing topic. We agree that conformational\nmovements associated with different stoichiometric interactions between CCM2 and KRIT1 may\nwell be functionally and mechanistically interesting. Our hunch is that structural assessments of\nconformational lability, potentially in the presence of MEKK3, may provide insights into both this\ncomment and the reviewer’s comment number 2. However, these are likely multi-year Ph.D.\nprojects and outside the scope of the current manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed the points raised in the reviews in a thoughtful and interesting\nmanner. I think the paper addresses key points that are of great interest to the field and bring\ntogether many productive years of study by many inveestigators approaching this disease\nthrough diverse methods.\n>RESPONSE. We greatly appreciate Reviewer 1’s insightful comments that helped to improve\nour study.\nReviewer #2 (Remarks to the Author):\nThe authors have sufficiently addressed the concerns of the reviewers. The current manuscript\nprovides solid evidence for the author's conclusions that two CCM2 proteins interact with a\nsingle KRIT1 molecule to suppress KLF4 expession. This work will be of wide interest and\nshould be accepted for publication.\n>RESPONSE. We thank Reviewer 2 for their thoughtful queries that helped to improve our\nstudy.\nReviewer #3 (Remarks to the Author):\nPublication in a journal of this caliber requires that the corresponding author adequately address\nall reviewer comments. In this review process, while some of my comments were addressed,\nthe authors did not sufficiently respond to the key questions that are essential to the scientific\nmerit of the study. Consequently, I cannot recommend this manuscript for publication.\n>RESPONSE. We thank Reviewer 3 for acknowledging that some of their comments were\naddressed and regret that additional points remain unresolved. Nonetheless, we believe our\nmanuscript makes a valuable contribution to the field and this conclusion is supported by the\ncomments from reviewers 1 and 2. We appreciate Reviewer 3’s comments in the original round\nof review which helped us improve our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the points raised in the reviews in a thoughtful and interesting manner. I think the paper addresses key points that are of great interest to the field and bring together many productive years of study by many inveestigators approaching this disease through diverse methods.", "reviewer_2": "The authors have sufficiently addressed the concerns of the reviewers. The current manuscript provides solid evidence for the author's conclusions that two CCM2 proteins interact with a single KRIT1 molecule to suppress KLF4 expession. This work will be of wide interest and should be accepted for publication.", "reviewer_3": "Publication in a journal of this caliber requires that the corresponding author adequately address all reviewer comments. In this review process, while some of my comments were addressed, the authors did not sufficiently respond to the key questions that are essential to the scientific merit of the study. Consequently, I cannot recommend this manuscript for publication."}, "author_response": ""}]

Decision letter