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

Abstract

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI‐MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the γ chain of the FcεRI‐receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 °C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of ∼9 kcal mol−1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI‐MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two‐step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal‐transduction processes.

Insights into the allosteric regulation of Syk association with receptor ITAM, a multi-state equilibrium

“…The apparent equilibrium dissociation constant of tSH2 and ITP is expected to be substantially lower than the limits achieved here. Reported values for the apparent K d are ~0.001 to 30 nM from other methods, 11–16 which is higher affinity than can be determined by direct NMR measurements. 41 …”

“…18 The conformational effect of phosphorylation is of considerably greater magnitude than the flexibility previously described for unphosphorylated Syk. 16 It was suggested that the allosteric inhibition of the Syk-receptor interaction by IA phosphorylation was through a conformational change in domain structure that switched the bifunctional high-affinity binding across two SH2 domains to a monofunctional lower-affinity binding that involved only one SH2 domain. 18 Thus, linker A phosphorylation was proposed to restrict the separation and relative orientation of two SH2 domains in a manner that no longer appropriately positioned the two phosphotyrosine binding sites to allow bifunctional binding, and therefore binding is by a single pYXX(I/L) cassette and the affinity for ITAM is reduced to a value comparable to a single SH2 domain.…”

Nanoparticle delivery of a peptide targeting EGFR signaling

“…EV peptide inhibited STATb phosphorylation far better than the Y845 sequence (Figure 1a). This result is not surprising; the natural Y845 domain may assume a favorable conformation only when the sequence is constrained by the context flanking sequences [30–31]. Although EV was active in inhibiting the phosphorylation of STAT5b, the peptide affinity was not particularly high (IC 50 = 2.5 μM).…”

Thermodynamics of binding interactions in the rational drug design process

“…This can lead to significant improvements in binding affinity. Of course, even where this rigidity is built into the small molecule, the entropic advantage may not always be realised as decreases in protein flexibility may still dominate [58] . The free energy, of course, is composed of the enthalpy and entropy terms and the different types of interaction often have individual thermodynamic signatures ( Figure 4 ).…”