Surface Plasmon Resonance Thermodynamic and Kinetic Analysis as a Strategic Tool in Drug Design. Distinct Ways for Phosphopeptides to Plug into Src- and Grb2 SH2 Domains

Abstract

Thermodynamic and kinetic studies of biomolecular interactions give insight into specificity of molecular recognition processes and advance rational drug design. Binding of phosphotyrosine (pY)-containing peptides to Src-and Grb2-SH2 domains was investigated using a surface plasmon resonance (SPR)-based method. This SPR assay yielded thermodynamic binding constants in solution, and the kinetic information contained in the SPR signal allowed kinetic analysis, which demonstrated distinct ways for pY ligands to interact with the SH2 domains. The results for binding to Src SH2 were consistent with sequestration of water molecules in the interface of the pYEEI peptide/Src SH2 complex. The results for a pYVNV peptide binding to Grb2 SH2 suggested a conformational change for Grb2 SH2 upon binding, which is not observed for Src SH2. Binding of a cyclic construct, allowing the pYVNV sequence in the bound conformation, did not have the expected entropy advantage. The results suggest an alternative binding mode for this construct, with the hydrophobic ring-closing part interacting with the protein. In all cases, except for full-length Grb2 protein, the affinity for the immobilized peptide at the SPR sensor and in solution was identical. This study demonstrates that SPR thermodynamic and kinetic analysis is a useful strategic tool in drug design.

Structural and energetic aspects of Grb2-SH2 domain-swapping

“…However, this increase in the entropy of binding is not sufficient to override the loss in enthalpy of binding. It is perhaps noteworthy that the dissociation constant reported herein for the binding of tripeptide Ac-pYVN to monomeric Grb2-SH2 (K d = 1.6 M) is somewhat greater than that reported for Ac-PSpYVNVQN-NH 2 (K d = ~0.3 M) [29] and comparable to the ELSIA-derived IC 50 value (K d = 4.3 M) for Ac-pTyr-Val-Asn-NH 2 reported by Garcia-Echeverria and colleagues [30].…”

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

“…Here we characterize the binding of a flexible diphosphorylated ITAM peptide and a rigid ITAM-derived ligand to the Syk tSH2 domain by thermodynamic and kinetic analysis by SPR, as well as by studying effects on protein dynamics with ESI-MS. From previous work by us and by others it appeared that such an SPR approach can give reliable thermodynamic data. [37,38] The affinity of Syk tSH2 for g-ITAM in solution was found to be identical to that for immobilized g-ITAM (1) at the SPR sensor surface. This has been observed previously for single SH2 domains, [38] and indicates that the affinity is not influenced by additional effects of the sensor chip.…”

“…[37,38] The affinity of Syk tSH2 for g-ITAM in solution was found to be identical to that for immobilized g-ITAM (1) at the SPR sensor surface. This has been observed previously for single SH2 domains, [38] and indicates that the affinity is not influenced by additional effects of the sensor chip. Comparison of the thermodynamic binding parameters of the flexible native peptide 1 with those of the rigid ligand 2 ( Figure 3, Table 1) shows …”

“…This is in accordance with our observations. [38] The sequential two-step model proposed here is different from the model proposed by Grucza et al for binding of eITAM to Syk tSH2. [26] Their model includes an equilibrium between two distinct conformations in the unbound state.…”

“…[42] With the SPR method used here we have indeed found good agreement between DH 0 values from both methods. [38] …”