Sam domains are small protein modules that act in several biological events and can form homotypic or heterotypic associations [20
]. The lipid phosphatase Ship2 [5
] contains a Sam domain at its C-terminus. Binding partners of this Sam domain have been only recently reported and consist of the Sam domain from the EphA2 receptor [11
] and the Sam domain from the PI3K effector protein Arap3 [3
]. The NMR solution structure of Ship2-Sam (pdb code: 2K4P
) has been recently determined in our laboratory [10
]. We have also investigated the interaction between Ship2-Sam and EphA2-Sam by means of ITC and NMR chemical shift perturbation studies [10
]. ITC data have shown that the Sam domain of Ship2 binds to the Sam domain of the EphA2 receptor with a dissociation constant Kd
= 0.75 ± 0.12 μM and a 1:1 binding stoichoimetry [10
To gain additional insights into the structural determinants characteristic of the Sam-Sam interactions which involve Ship2, we have now performed similar structural and binding studies on the association of Ship2-Sam with Arap3-Sam. ITC data by Ramajjmaker et al. [3
] have already reported a Kd
value of about 100 nM and a single binding site model for this interaction.
The solution structure of Arap3-Sam consists of a small five helix bundle and represents a classical Sam-domain fold (Figure ). In fact, Arap3-Sam presents highest sequence homology with the Sam domain of Arap2 (pdb code: 1X40
, Riken Structural Genomics Initiative) as shown by a blastp [22
] search of the Arap3-Sam primary sequence versus
the Protein Data Bank (pdb) database [23
]. Sequence similarities with Ship2-Sam (pdb code: 2K4P
]) and EphA2-Sam (pdb code: 2E8N
, Riken Structural Genomics Initiative) are also relatively high (49% and 58% respectively) (Figure , upper panel). Previous studies have already reported that like Ship2-Sam, Arap3-Sam does not have a strong propensity to self-associate and prefers to be involved in heterotypic interactions [3
]. Our 15
N R1 and R2 relaxation measurements together with analytical ultracentrifugation studies, further validate these findings.
Chemical shift mapping studies indicate that the interaction surface of Ship2-Sam for Arap3-Sam is mainly made up of the central regions of the protein (Figure ). This is the same area that we identified as responsible for the binding of Ship2-Sam to EphA2-Sam (Figure ). The Shiptide, a 22 residue long Ship2-Sam peptide, representing the minimal Ship2-Sam region capable of binding to EphA2-Sam, retains some ability to bind Arap3-Sam as shown by ITC (Figure ). In the case of Arap3-Sam/Shiptide interaction, enthalpic contributions are responsible for ~44% of the free energy of binding (ΔH
= -2.6 kcal/mol, ΔG
= -5.9 kcal/mol) whereas for EphA2-Sam/Shiptide interaction [10
] the enthalpy contributes only ~22% to the free energy of binding (ΔH
= -1.4 kcal/mol, ΔG
= -6.5 kcal/mol). Thus, in the Arap3-Sam/Shiptide complex hydrogen bonding and electrostatic interactions are more predominant.
In addition, NMR displacement experiments clearly show that Arap3-Sam and EphA2-Sam compete for the same binding site on the surface of Ship2-Sam (Additional File 3
The binding area of Arap3-Sam for Ship2-Sam is primarily made up of the C-terminal α 5 helix and adjacent loop regions (Figure ). Again, the location of this binding site closely resembles the interaction surface of EphA2-Sam for Ship2-Sam. From these binding data, we conclude that Ship2-Sam and Arap3-Sam most likely interact by using the Mid-Loop (ML)/End-Helix (EH) Model that is common among Sam-Sam associations [8
] and where Ship2-Sam and Arap3-Sam are providing the Mid-Loop and End-Helix interfaces respectively (Figure ). The same interaction mode has been previously proposed by us for the interaction between Ship2-Sam and EphA2-Sam.
A model of the Ship2-Sam/Arap3-Sam complex was generated with the software Haddock 1.3 [18
] by using chemical shift perturbation data (Figure ). The best scoring solution represents well the ML/EH topology that is present in other experimental structure of Sam-Sam complexes [8
The binding site of Ship2-Sam for Arap3-Sam contains many negatively charged residues, while the EH interface of Arap3-Sam includes several positively charged amino acids. As a consequence, this model appears largely stabilized by electrostatic interactions (Figure ). In fact, by destroying some of these interactions through simultaneous mutation of the positively charged Arap3-Sam residues H37, R77 and R80 to aspartic acids, the binding to Ship2-Sam is abolished or at least highly attenuated as shown by NMR binding data (Additional File 2
A very similar interaction model has been obtained by us for the Ship2-Sam/EphA2-Sam association, by means of docking procedures [10
] (Figure ). The pattern of interactions at the dimer interface is analogous in the two complexes (Figure ). It is worth noting that in the Ship2-Sam/EphA2-Sam model the EphA2-Sam residue Y81 may form a stacking π-π interaction with the Ship2-Sam residue F55 that can be replaced by the cation-π interaction between the Arap3-Sam residue R80 and F55 of Ship2 in the complex Ship2-Sam/Arap3-Sam. Furthermore, in the Ship2-Sam/Arap3-Sam complex H61 of Arap3-Sam could provide an additional electrostatic interaction with D63 of Ship2-Sam that is not permitted in the Ship2-Sam/EphA2-Sam dimer (Figure ). These observations may reflect the relatively stronger binding observed between Arap3-Sam and Ship2-Sam (Kd
= ~0.1 μM [3
]) compared to the binding of EphA2-Sam to Ship2-Sam (Kd
= 0.75 ± 0.12 μM) as well as the better Haddock score for the Arap3-Sam/Ship2-Sam model (Figure , lower panel).