Homologous ligands accommodated by discrete conformations of a buried cavity

Abstract

Conformational change in protein–ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

“…Remarkably, there is a high degree of cavity plasticity, exceeding what might be expected from previous crystallographic studies (18,19), that involves residues, metastates, and conformations beyond the discrete crystallographic states observed for L99A bound to a congeneric series of benzene-related compounds (24,30). Further, the number of rotamers observed for buried L99A residues exceeds that seen in the several-hundred-microsecond simulations of other protein cores, such as ubiquitin, RNaseH, and b-lactamase (56).…”

“…L99A is a model system for studying ligand binding to buried protein cavities and protein excited states, and the conformational changes that govern these phenomena have been enigmatic despite nearly 25 years of experimental study (5,6,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Experimental studies of the L99A mutant of T4 lysozyme have focused on defining the structures of and transition times between the ground state, the excited state, and ligand-bound states.…”

“…Initial crystal structures of the L99A mutant indicate that the~40 Å 3 cavity in the C-terminal domain of wild-type protein expands to an~150 Å 3 cavity (26), a volume capable of accommodating substituted benzenes and noble gases (20,24,(27)(28)(29)(30). O 2 is simulated to bind and escape the buried cavity through openings between the D, E, G, H, and J helices (31), whereas binding of substituted benzenes is accompanied by discrete rearrangements in the F and G helices (30). Substantial rearrangements also occur in these helices in an excited state that comprises~3% of the protein ensemble at room temperature (5,25).…”

Sensitivity in Binding Free Energies Due to Protein Reorganization

“…In this study, we model recent experimental results that show the progressive opening of the binding pocket in response to a series of homologous ligands. 1 Even while using enhanced sampling techniques, we demonstrate that the predicted relative binding free energies (RBFE) are sensitive to the initial protein conformational state. Particularly, we highlight the importance of sufficient sampling of protein conformational changes and demonstrate how inclusion of three key protein residues in the ‘hot’ region of the FEP/REST simulation improves the sampling and resolves this sensitivity.…”

“…Regardless, it is clear that protein-ligand binding can be highly complex, and multiple stable protein conformations may be populated even for the same ligand. 1,26,27 Thus, there is a great need to understand how protein conformational changes and the choice of starting protein conformation may affect the reliability of free energy predictions.…”