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Page 6 of 23 Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34

CCM1 is independent of binding to RAP1. HEG1 interacts with the hydrophobic pocket between F1 and F3
subdomain. The interaction between HEG1 and CCM1 is trifold: F1 subdomain through polar interactions,
F3 subdomain through hydrophobic interaction, and α2A helix between F1 and F3 subdomain. Analysis of
electron density shows that the C-terminal dipeptide on HEG1 of tyrosine-phenylalanine is critical for their
binding. This binding is not in the category of the classical PH (F3 subdomain) - NPxY motif interaction. In
fact, the binding pocket is similar to the inositol phosphate binding site in the PH domain . Similar to the
[75]
binding of RAP1, the binding of HEG1 doesn’t illicit any conformational change in the FERM domain .
[43]
The binding of CCM1 with HEG1 and RAP1 is essential for appropriate cardiovascular development,
and disruption of these interactions was shown to cause the phenotype of CCM . Heg1-null zebrafishes
[71]
manifest with the same cardiovascular phenotype as Ccm1 null mutants . Infusion of CCM1 mutants
[76]
lacking the binding ability to RAP1 and HEG1 into Ccm1 null zebrafish could not reverse the cardiovascular
phenotype [71,76] , indicating the essential role of the interaction.

Multiple NPXY motifs in CCM1 protein
CCM1 binds to ICAP1α and acts a competitive inhibitor of ICAP1α and β1-integrin interactions[20,68].
ICAP1α contains a PTB domain that binds to β1-integrin and modulate β1-integrin mediated cellular
function [15,23-25,54] . Mutagenesis of the T778 and V790 or N792 and Y795 of the cytoplasmic tails of β1-integrin
prevents binding to ICAP1α. However, the exact consequence of this interaction is not fully agreed upon.
The overwhelming theory is that CCM1 acts to sequester ICAP1α resulting in increased levels of β1-integrin
activation because ICAP1α is a potent repressor of β1-integrins [15,23-25,54] . However, one study suggests that
CCM1 interaction with ICAP1α stabilizes ICAP1α, therefore increasing β1-integrin activation. This effect
is profound when low levels of CCM1 results in paradoxically increased β1-integrin activation.However,
appropriate amount of β1-integrin is necessary for development of vascular sinusoids , cell cycle [78,79] , and
[77]
bone development [80,81] . ICAP1α PTB domain is a DAB-like PTB domain where the interaction between
ICAP1α and CCM1 is independent of the phosphorylation status of the NPXY motif. Binding of CCM1
or β1-integrin to ICAP1α doesn’t result in any structural changes . CCM1 binding to ICAP1α through a
[82]
bidentate interaction with the ICAP1α PTB domain. This interaction is identical to ICAP1α and β1-integrin
interaction thereby resulting in competitive inhibition [15,23-25] . CCM1 utilizes the first NPXY motif and a RR
region to bind to ICAP1α PTB domain . The RR region is a novel site that binds to the N-terminus of NPXY
[83]
motif. This N- terminal region adopts an α-helix confirmation that binds to the loop between β1 and β2 and
well as β5 and β6 and α1 helix of the PTB domain . The interaction between the PTB domain and the RR
[82]
site is mediated by the conserved arginine residues on the CCM1 (R179 and R185), binding to polar side
chains such as the carbonyl groups of D146, D93, and Q96 on ICAP1α. The interaction of the PTB domain
and the NPxY motif is mediated by the interaction of N192 and Y195 on CCM1 to the ICAP1α binding site
of PTB domain, L135, I138, I139, and C184. Mutation in the NPXY binding sites on ICAP1α inhibits binding
to cytoplasmic tail of β1 integrin. However, mutation in the RR binding sites (D146, D93, and Q96) did not
affect binding with cytoplasmic tail of β1 integrin. Therefore, unlike CCM1-ICAP1α interaction, the binding
between ICAP1α and β1-integrin is not a bidentate interaction .
[54]
CCM1 binds to CCM2 and SNX17 through the utilization of the second and third NPXY motifs [18,19,59,84] .
Biochemical studies demonstrate that CCM2 utilizes its PTB domain to bind to the third NPXY motif of
CCM1. Several leucine residues in CCM2 (L113, L115, L155, L198, and L213) are paramount for adequate
binding to CCM1. Several residues downstreaming to the NPXY motif such as V244 and V248 are also
important. Even a conservative mutation like V244L was found to significantly decrease binding. An X-ray
crystallography of CCM2 with the third NPxY motif of CCM1 was determined. However, Co-IP shows that
both the second and third NPxY motifs are required to bind to CCM1 . Additionally, biochemical studies
[18]
do not show an increased affinity with a construct containing both the second and third NPxY motifs over
just the third NPxY motif .
[59]