Binding is not enough: flexibility is needed for photocrosslinking of Lck kinase by benzophenone photoligands

Abstract

Benzophenone photophores are employed widely for photoaffinity-labeling studies. Photolabeling with benzophenone, however, is hardly a routine experiment. Even when a photoprobe binds to its target, photocrosslinking does not necessarily occur. This is because photolabeling by benzophenone is affected by many factors other than target-binding, such as conformational flexibility of photoligand. Despite the widespread recognition of such complications, there has been no systematic study to assess the relative importance of individual factors that can affect photolabeling efficiency. In order to gain an insight into this problem, we conducted a structure-activity relationship (SAR) study of benzophenone photoligands for Lck kinase, in which photoligands with varying target-binding affinity and conformational flexibility were compared. The study found that binding-affinity, as indicated by kinase inhibitory potency, did not correlate with photolabeling efficiency. Instead, conformational flexibility was found to be the determining factor for efficient photolabeling by our photoligands. Implication of the current findings, in particular, with regard to selection and optimization of benzophenone photoligands, is discussed.

Figure 1

The model of the Lck-Ligand complex. The central Gly between PNA-adenine and Bpa is highlighted by the yellow arrow. For clarity the biotin moiety is removed in this figure.

Figure 2

Structures of newly synthesized Lck photoligands. The “Lck-targeting motif” is highlighted by the blue bars in 1 (Gly). The glycine between Bpa and PNA-adenine in 1 (shown in red) is replaced with different amino acid residues. See text for more details.

Figure 3

The IC₅₀ values of Lck photoligands. Lck kinase reactions in the presence and absence of photoligands were carried out on a 384-well plate. Following the incubation, residual ATP in each well was quantified with Promega Kinase-Glo® Plus Luminescent Kinase Assay. See the Experimental section for more details.

Figure 4

Photoaffinity-labeling study of newly synthesized Lck ligands. (a) The gel image of Lck tagged with different concentrations of 1, and the resulting titration curve, from which EC₅₀ was estimated. (b) EC₅₀ values of all photoligands: triplicate experiments (n=3) were made for each data point.

Figure 5

Stability of photoligands under UV. Ligand solutions were irradiated under a UV-A lamp (λ_max 350 nm). At different time points (0, 10, and 30 min), aliquots were taken and the amounts of intact ligand was quantified by HPLC (UV 280 nm). See the Methods and Materials section for more details.