A dry ligand-binding cavity in a solvated protein

Abstract

Ligands usually bind to proteins by displacing water from the binding site. The affinity and kinetics of binding therefore depend on the hydration characteristics of the site. Here, we show that the extreme case of a completely dehydrated free binding site is realized for the large nonpolar binding cavity in bovine ␤-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water 17 O and 2 H magnetic relaxation dispersion (MRD), 13 C NMR spectroscopy, molecular dynamics simulations, and free energy calculations to establish the absence of water from the binding cavity. Whereas carbon nanotubes of the same diameter are filled by a hydrogenbonded water chain, the MRD data show that the binding pore in the apo protein is either empty or contains water molecules with subnanosecond residence times. However, the latter possibility is ruled out by the computed hydration free energies, so we conclude that the 315 Å 3 binding pore is completely empty. The apo protein is thus poised for efficient binding of fatty acids and other nonpolar ligands. The qualitatively different hydration of the ␤-lactoglobulin pore and carbon nanotubes is caused by subtle differences in water-wall interactions and water entropy.␤-lactoglobulin ͉ free energy simulation ͉ hydrophobic hydration ͉ magnetic relaxation dispersion G lobular proteins are stabilized by dense atomic packing and by water exclusion from the nonpolar core region. However, the packing density is not uniform and a typical protein contains approximately four cavities per 100 residues of sufficient size to accommodate at least one water molecule (1). Small nonpolar cavities are usually empty and can be regarded as packing defects, whereas small polar cavities tend to be occupied by structural water molecules that stabilize the folded protein by H-bonding to otherwise unsatisfied peptide partners (2). Large cavities are frequently linked to protein functions, such as ligand binding and transport, membrane translocation, and enzyme catalysis. Some of these large cavities are lined exclusively or predominantly by nonpolar sidechains. The question whether such large nonpolar cavities are empty or contain water molecules is of fundamental biological importance (3-8).The principal tool of structural biology, x-ray crystallography, cannot easily resolve this issue. There are three potential problems. (i) Because water molecules interact only weakly with the nonpolar cavity walls, they tend to be positionally disordered. As a result of such delocalization, the electron density of the water molecules may fall below the detection limit. (ii) At a typical resolution limit of Ϸ2 Å, a contaminating nonpolar ligand may be misinterpreted as a water cluster (9, 10). (iii) For structures determined at cryogenic temperatures, the hydration status of the cavity may be altered by re-equilibration during the flash cooling process (11).In favorable cases, water molecules in nonpolar protein cavities can be inferred from intermolecular nuclear O...

Capturing LTA 4 hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB 4 synthesis

“…3B and 4A and Movie S2). In this capacity, Y378 becomes a molecular gate for entry of LTA 4 into the hydrophobic tunnel and, simultaneously, prevents access of water molecules that could otherwise interfere with subsequent binding of the lipid substrate, as previously reported for the soluble bovine β-lactoglobulin (15). Of note, we could not find any structural obstacles for peptide substrate binding and turnover in the active site during lid displacement (Fig.…”

Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor

“…Small pockets (Ͻ25 Å 3 ) are not unusual, but lack well-characterized functional roles and are thought to be caused by imperfect side-chain packing in the protein core. Larger, single cavities are much less common, including the extensively hydrated 365-Å 3 internal cavity observed in the 55 kDa Brevibacterium sterolicum cholesterol oxidase (34) and the 315-to 450-Å 3 dry or fractionally hydrated cavities observed in several odorant binding proteins (35)(36)(37). Critically, all of these large internal cavities, which are comparable to the 290 Å 3 cavity we observed in HIF2␣ PAS-B, are present in the substrate-or ligand-free forms of these proteins.…”

Solvent fluctuations in hydrophobic cavity–ligand binding kinetics

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