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Page 8 of 23 Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34

cardiomegaly. This phenotype is seen to a milder extent in Ccm1, Ccm2, and Heg1 null mutants. The effect
of Ccm2l is more pronounced in the heart than large vessels. In fact, there is partial phenotypic rescue with
Ccm2 over-induction in Ccm2l-null zebrafish. Therefore, it is concluded that Ccm2l acts in the Heg-Ccm
pathway . It is still unclear whether CCM2L is involved in the pathogenesis of CCM .
[93]
[26]
CCM3 dimerization
The N-terminus of one CCM3 can bind to another CCM3 in the native state, driven by identical hydrophobic
[94]
residues, L44, A47, I66, and L67 . An important set of binding partners of CCM3 are GCKIII kinases
such as Mst4 and Stk25. These kinases contain an N-terminal catalytic domain and C-terminal regulatory
domain. The C-terminal regulatory domain of GCKIII can bind to dimerization domain in CCM3. The
tertiary structure of the C-terminus of GCKIII is similar to the N-terminus of CCM3 and can compete with
and replace a CCM3 in the CCM3 homodimer. These kinases adopt an independent V-shaped domain as
well. Each side of the V in the GCKIII protein is made up of several α-helices. Mutations in the hydrophobic
residues that removed CCM3 dimerization effectively also inhibit GCKIII and CCM3 binding . However,
[95]
CCM3 has higher binding affinity to GCKIII kinases over CCM3 homodimer. There exists a long linker
peptide in CCM3 between α3 and α4 helix along the CCM3 protein. In the homodimer state, this region folds
into α-helix and merges with α4 forming an extended helix. This extended helix is stabilized by hydrophobic
residues found in the antiparallel α1 helix in the partner CCM3 within the homodimer. Hydrophobic residues
(V72, F76, L80, M83) in the extended α4 helix interact with hydrophobic residues (V25, A24, P21, M20, V18,
and M17) in α1 helix of partner CCM3. This stabilizing interaction doesn’t occur in the GCKIII-CCM3
complex, because the corresponding α1 helix in GCKIII doesn’t contain any hydrophobic residues. In the
interaction between CCM3-GCKIII, the linker region between α3 and α4 helices in CCM3 is less structured
and able to adopt a flexible conformation. This allows hydrophobic residues (F76, L80, and M83) in α4 helix
in CCM3 to fall into a hydrophobic pocket formed by α1 and α3 helix in CCM3 through intra-molecular
binding. This twisting of the CCM3 causes in the full N-terminal face of CCM3 to interact with the full
C-terminal face of GCKIII, which doesn’t occur in the homodimer of CCM3 due to the inherent bend in
the CCM3 tertiary structure [63,96] . Therefore, there is a larger binding area between CCM3 and GCKIII. This
[95]
larger-interface interaction present results in a much higher binding affinity .
Knockout models of Stk24 and Stk25 caused cardiovascular disease similar to that observed in CCM3
knockout models . STK25, a serine/threonine kinase, controls RhoA activation, which is a GTPase. Loss of
[97]
RhoA results in stress fiber formation . It has been well documented that CCM phenotype causes increased
[98]
stress fiber formation; however several reports contradicted this finding [99,100] . Increase RhoA activation
leads to increased ROCK. ROCK is another serine/threonine kinase that phosphorylates several proteins:
myosin light chain, MLC phosphatase, and LIM kinase. MLC phosphatase decreases the cross linking of
myosin and actin, the source of fiber contractility. Phosphorylation by ROCK inhibits MLC phosphatase.
In contrast, ROCK dependent phosphorylation of LIM kinase results in activation. Active LIM kinase
catalyzes phosphorylation of cofilin, which inhibits cofilin activity that regulates actin depolymerization.
Both pathways, LIM kinase and MLC phosphatase, lead to stress fiber formation. ROCK inhibition results
in regression of stress fiber . Increases in ROCK activity have been recorded in CCM lesions. Interestingly,
[26]
increases in ROCK activity have been seen in histologically normal blood vessels in CCM1 deficient mice,
suggesting possible involvement of ROCK signaling in the pathogenesis of CCM lesions .
[101]

CCM3 C-terminal FAT-H domain
CCM3 contains a FAT-H domain at the C- terminus that is used to bind to CCM2. The surface of the domain
contains a hydrophobic patch termed hydrophobic patch 1 (HP1), which is found between α7 and α8 helix.
This is the site for binding various proteins such as CCM2 , striatins , and paxillin . For interaction of
[61]
[63]
[102]
both stratins and paxillin, CCM3 recognizes LD motifs that adopt helical structures. Compared to CCM2-
CCM3 complex, CCM3-paxillin complex has a smaller surface area, the LD motif is smaller (~ 2 turns),
and the LD motif helix is less parallel to α7 helix in CCM3 . Paxillin is known to bind to FAT-H domains
[91]