Protein conformer selection by ligand binding observed with crystallography

Abstract

A large-scale movement between "closed' and "open" conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of TrpI9' in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop P r~'~~-A s n '~~ and exposing TrpI9' to the protein surface. Kinetic measurements are consistent with a two-step binding mechanism in which the rate-limiting step is a transition of the protein to the open state, which then binds the ligand. This large-scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild-type protein is in the open form in solution at any given time.Keywords: artificial cavity; cytochrome c peroxidase; kinetics; ligand binding; loop movement; protein crystallography; site-directed mutagenesis Much evidence exists that proteins exhibit considerable flexibility. Ligands are often found in the center of proteins with no observable path to the surface, suggesting that the protein must transiently unfold in order for the ligand to enter. Examples include myoglobin and hemoglobin oxygen binding to the heme (Karplus & Petsko, 1990), fatty acid binding to fatty acid binding protein (LaLonde et al., 1994), retinol binding to retinol binding protein (Zanotti et al., 1993), and ligand binding to artificial cavities introduced into proteins (Eriksson et al., 1992;Fitzgerald et al., 1994Fitzgerald et al., , 1996Feher et al., 1996). Flexible surface-loops in protein structure are thought to play an important role in substrate binding and molecular recognition. Crystal structures have shown that, for a number of enzymes and antibodies, substrate and/or antigen binding causes a conformational change of a surface-loop either from an "open" to a "closed" form to partially cover the bound substrate (Joseph et al., 1990;Lolis & Petsko, 1990;Derewenda et al., 1992;Jia et al., 1995), or from a "partially closed' to "open" form, resulting in an induced fit for the antigen or ligand binding (Rini et al., 1992;Concha et al., 1993). These conformational changes have been observed directly by NMR, and the relevance of these motions to dihydrofolate reductase function has been demonstrated (Epstein et al., 1995). All of these examples reveal that Reprint requests to: Duncan E. McRee, Department of Molecular Biology"MB8, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037; e-mail: dem@scripps.edu.Abbreviations: CCP, cytochrome c peroxidase; wtCCP, wild-type CCP; cyt c, cytochrome c; DMI', 1,2-dimethylimidazolium; 2a5mT+, 2-amino-5-methylthiazolium; MPD, 2-methyl-2,4-pentanediol.proteins switch between conformations and that these changes are often key to protein function.Here we trap a large conformational change of a protein loop by binding an exogenous ligand to the protein and directly visualizing the structure...

Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L)

“…Between pH 5.5 and 6.5, Fig. 8A, $k_s^{normal\mathit{obs}}$ decreases with increasing concentration of MIM similar to the concentration dependence of 1,2-dimethylimidazole binding within the Trp-191 cavity of CcP [20]. Between pH 4.1 and 5.0 and at pH 7.0 and 7.5, the value of $k_s^{normal\mathit{obs}}$ attains a minimum value at intermediate values of MIM and then increases with increasing ligand concentration, Figs.…”

“…In the reported studies, imidazole does not bind to the heme iron but rather binds within a cavity formed in the proximal heme pocket by movement of Trp-191 from the interior of the protein to the surface [19,20]. The movement of Trp-191 creates two conformations of the enzyme, with native CcP existing in a “closed” conformation, with Trp-191 in van der Waals contact of the heme, 96% of the time and in an “open” conformation, with Trp-191 exposed to the solvent 4% of the time [20]. The conformational equilibrium is due to the large scale movement of residues 190 to 195 that comprise a loop on the surface of CcP.…”

Conformational behavior of ionic self‐complementary peptides

“…Alpha-helices and b-sheets are generally believed to be stable structural units after they have formed in folded proteins~Pauling, 1960; Schulz & Schirmer, 1979;Creighton, 1993!. Recent analyses using high-speed computer simulations and using a variety of experimental approaches suggest that protein structures are dynamic and they can undergo structural changes to accommodate their biological function~Boutonnet et al, 1995;Cao et al, 1998;Kurzynski, 1998;Reddy et al, 1998;Yon et al, 1998!. These include receptor-ligand interactions, enzyme catalysis and protein interactions with other molecules, including other proteins, nucleic acids, small molecules, cofactors, metal ions, neurotransmitters, and other substrates~Colson et al, 1998;Haouz et al, 1998;Rigney et al, 1998!.…”

Exploiting Ligand and Receptor Adaptability in Rational Drug Design Using Dynamics and Structure-Based Strategies

“…The exact manner by which these changes occur in solution are likely via an ensemble of rapidly interconverting structures, and the complexation with a rigid peptidyl ligand results in a restriction of the flexibility of the protein active-site. In this respect, the process bears elements of protein conformer selection [28]. In other words, the peptide guides the reorganization of the protein around it after association.…”