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PDBsum entry 4wci
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Signaling protein
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PDB id
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4wci
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References listed in PDB file
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Key reference
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Title
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Differential recognition preferences of the three src homology 3 (sh3) domains from the adaptor cd2-Associated protein (cd2ap) and direct association with ras and rab interactor 3 (rin3).
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Authors
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E.Rouka,
P.C.Simister,
M.Janning,
J.Kumbrink,
T.Konstantinou,
J.R.Muniz,
D.Joshi,
N.O'Reilly,
R.Volkmer,
B.Ritter,
S.Knapp,
F.Von delft,
K.H.Kirsch,
S.M.Feller.
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Ref.
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J Biol Chem, 2015,
290,
25275-25292.
[DOI no: ]
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PubMed id
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Abstract
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CD2AP is an adaptor protein involved in membrane trafficking, with essential
roles in maintaining podocyte function within the kidney glomerulus. CD2AP
contains three Src homology 3 (SH3) domains that mediate multiple
protein-protein interactions. However, a detailed comparison of the molecular
binding preferences of each SH3 remained unexplored, as well as the discovery of
novel interactors. Thus, we studied the binding properties of each SH3 domain to
the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a
peptide array screen based on the recognition motif PxPxPR and identified 40
known or novel candidate binding proteins, such as RIN3, a RAB5-activating
guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound
with similar characteristics and specificities, whereas the SH3-3 domain bound
more weakly to most peptide ligands tested yet recognized an unusually extended
sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays
revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3
appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to
RAB5a-positive early endosomes via these interaction sites. Permutation arrays
and isothermal titration calorimetry data showed that the preferred binding
motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å)
of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2,
respectively, indicated that another extended motif is relevant in epitope 2. In
conclusion, we have discovered novel interaction candidates for CD2AP and
characterized subtle yet significant differences in the recognition preferences
of its three SH3 domains for c-CBL, ALIX, and RIN3.
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