Specificity of ligand binding in a buried nonpolar cavity of T4 lysozyme: Linkage of dynamics and structural plasticity

Abstract

To better understand the role of shape complementarity in ligand binding and protein core interactions, the structures have been determined of a set of ligands bound within a cavity-containing mutant of T4 lysozyme. The interior cavity is seen to consist of two parts that respond very differently to the binding of ligands. First, there is a relatively rigid region that does not relax significantly upon binding any ligand. Second, there is a more flexible region that moves to various extents in response to binding the different ligands. The part of the binding site that remains rigid is characterized by low temperature factors and strong protection from hydrogen exchange. This part of the site appears to be primarily responsible for discriminating between ligands of different shape (i.e., for determining specificity). The more flexible region, characterized by relatively high temperature factors and weak protection from hydrogen exchange, allows some promiscuity in binding by undergoing variable amounts of deformation at essentially the same energetic cost. This linkage between the dynamic information represented by crystallographic temperature factors and hydrogen-exchange behavior on the one hand, and structural plasticity in response to ligand binding on the other hand, suggests a way to improve our understanding of steric interactions in protein cores and protein-ligand binding sites. Ligand design and packing algorithms might take advantage of this information, requiring complementary interactions where the protein is rigid and allowing some overlap in regions where the protein is flexible.

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.…”

“…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).…”

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

“…The refined model suggests that backbone atoms within the helix move up to 2 8, (Fig. 15) interaction between Val 1 1 1 and Leu 84 is important in localizing helix F (Morton & Matthews, 1995). The observation that the helix Isoleucine 27 to alanine plus isoleucine 58 to alanine is substantially disordered on substituting Leu 84 with alanine When both Ile 27 and Ile 58 are simultaneously replaced by alasupports this assertion.…”

Protein–Ligand Binding Free Energies from Exhaustive Docking

“…The T4 lysozyme L99A mutant satisfies these criteria. 17 The mutation creates a nonpolar cavity that can accommodate a number of small molecules. The cavity has the added advantage that it is entirely buried and unsolvated.…”