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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 3 of 23

is around 88% . A recurrent CCM1 missense mutation (Q455X) is prevalent in the Hispanic population,
[40]
hence it is named common Hispanic mutation . It is present in high frequency in southwestern US . From
[7]
[41]
N-terminal to C-terminal, CCM1 protein contains a NUDIX domain, three NPxY/F motifs, Ankyrin repeat
domain, a FERM domain, nuclear export signal (residues 551-562) [18,25,42] and nuclear localization signals
(residues 46-51, 569-572) [23,42-44] .

CCM1 C-terminal FERM domain
Located at the C-terminus (residues 420-736), the FERM domain contains three subdomains (F1-F3). The F1
subdomain of the FERM domain folds into an ubiquitin-like fold, while the F2 subdomain folds into an acyl-
CoA binding fold. Several proteins are known binding partners to this region of CCM1 . The F3 subdomain
[45]
of FERM domain, a bona fide PH domain, binds to NPXY motifs. There have been several reports on this
type of interaction in CCM1 [19,46-48] . Some reports indicate that the C-terminal FERM domain binds to its
first NPXY motif intramolecularly, thereby allowing the CCM1 protein to adopt a closed conformation. They
further suggested that in the open conformation, the CCM1 binds to ICAP1α and localizes it to the cytoplasm
and to the nucleus. In the closed conformation, the protein’s binding site is inaccessible to ICAP1α and results
in increased CCM1 binding to microtubules through the NUDIX domain, retaining ICAP1α in the cell
membrane. In support of this theory, it was further suggested that cellular binding partners of CCM1 to the F1/
F2 subdomain of the FERM domain or NUDIX domain may drive this conformational change in CCM1 [47,48] .
However, new evidence showed that unlike most N-terminal FERM domain-containing proteins, CCM1
(containing a C-terminal FERM domain) does not seem to follow the auto-inhibition mechanism to undergo
an intramolecular folding. In fact, both dynamic light scattering (DLS) and native protein gel electrophoresis
showed an increased tendency of CCM1 to undergo CCM3 - like oligomerization through intermolecular
binding between F3 subdomain of FERM domain and 3 NPXY motifs among partnering CCM1 proteins over
intramolecular binding of F3 subdomain of FERM domain - NPXY motifs within CCM1 .
[19]

CCM1 ankyrin repeat domain
The ankyrin repeat domain (ARD) domain (residues 288 and 419) in CCM1 is composed of 4 ankyrin repeats.
This ARD packs onto the N-terminal side of the F1 subdomain of the FERM domain. Each repeat is identical
and composed of two α-helices joined by a β-hairpin. The binding site is between the α-helices. These four
Ankyrin repeats stack vertically into an “L”-shaped fold. The fourth repeat contains a tripeptide insert (G401,
N402, and N403) in the binding site stabilized by a conversed W404. This tryptophan residue sits in a highly
conserved hydrophobic pocket. X-ray crystallography showed that the ARD domain is bound tightly to
the FERM domain. This highly conserved interaction is particularly mediated by the convex surfaces of
Ankyrin repeats 2 and 3 in ARD domain and the β2 strand and α2 helix in F1 subdomain in the FERM
domain. This interaction is stabilized by 10-12 hydrogen bonds over 993 A2. The ARD domain was termed as
the F0 subdomain in the FERM domain due to its proximity to the FERM domain. The presence of another
domain in such as position has been found in other FERM domain - containing proteins, i.e., Talin and
Kindlin [49,50] . Many proteins such as RAP1 and HEG1 are able to bind to the FERM domain with little to no
structural change of the ARD domain required. The ARD in CCM1 is different from other ARD in proteins
such as DARPins due to its inability to bind to β-tubulin. CCM1 was suggested to be a tubulin binding
protein, however both structural studies and binding assays show that the ARD domain is not utilized for
this interaction [51,52] . Till now, there are no known binding partners to the ARD domain in CCM1.

Multiple NPXY motifs in CCM1 protein
CCM1 also contains 3 NPXY motifs (residues 192-195, NPAY; 231-234, NPLF; 250-253, NPYF) in the central
portion of the protein which provide important interactions with phosphotyrosine binding (PTB), PH,
FERM domains, etc. The first motif, which is the only one that can be phosphorylated, has a remarkably
strong binding affinity to ICAP1α [20,25] . NPXY motifs 2 and 3 can only bind to DAB-like PTB domains,
including CCM2 [18,19] .