Small Molecule Binding to an Artificially Created Cavity at the Active Site of Cytochrome c Peroxidase

Fitzgerald, Melissa M.; Churchill, Michael; McRee, Duncan E.; Goodin, David B.

doi:10.1021/bi00179a004

Cited by 103 publications

(167 citation statements)

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“…4 and SI Appendix, Fig. S8) (47). Affinities for 11 ligands were determined at least in duplicate and fit to a one-site binding model with R 2 values of at least 95%.…”

Section: Resultsmentioning

confidence: 99%

Testing inhomogeneous solvation theory in structure-based ligand discovery

Balius

Fischer

Stein

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Binding-site water is often displaced upon ligand recognition, but is commonly neglected in structure-based ligand discovery. Inhomogeneous solvation theory (IST) has become popular for treating this effect, but it has not been tested in controlled experiments at atomic resolution. To do so, we turned to a grid-based version of this method, GIST, readily implemented in molecular docking. Whereas the term only improves docking modestly in retrospective ligand enrichment, it could be added without disrupting performance. We thus turned to prospective docking of large libraries to investigate GIST's impact on ligand discovery, geometry, and water structure in a model cavity site well-suited to exploring these terms. Although top-ranked docked molecules with and without the GIST term often overlapped, many ligands were meaningfully prioritized or deprioritized; some of these were selected for testing. Experimentally, 13/14 molecules prioritized by GIST did bind, whereas none of the molecules that it deprioritized were observed to bind. Nine crystal complexes were determined. In six, the ligand geometry corresponded to that predicted by GIST, for one of these the pose without the GIST term was wrong, and three crystallographic poses differed from both predictions. Notably, in one structure, an ordered water molecule with a high GIST displacement penalty was observed to stay in place. Inclusion of this water-displacement term can substantially improve the hit rates and ligand geometries from docking screens, although the magnitude of its effects can be small and its impact in drug binding sites merits further controlled studies.water | inhomogeneous solvation theory | ligand discovery | structure-based drug design | docking T he treatment of receptor-bound water molecules, which are crucial for ligand recognition, is a widely recognized challenge in structure-based discovery (1-4). The more tightly bound a water in a site, the greater the penalty for its displacement upon ligand binding, ultimately leading to its retention and the adoption of ligand geometries that do not displace it. More problematic still is when a new bridging water mediates interactions between the ligand and the receptor. Because the energetics of bound water molecules have been challenging to calculate and bridging waters hard to anticipate, large-scale docking of chemical libraries has typically been conducted against artificially desolvated sites or has kept a handful of ordered water molecules that are treated as part of the site, based on structural precedence (5-8).Recently, several relatively fast approaches, pragmatic for early discovery, have been advanced to account for the differential displacement energies of bound water molecules (9-20), complementing more rigorous but computationally expensive approaches (18)(19)(20)(21)(22). Among the most popular of these has been inhomogeneous solvation theory (IST) (23)(24)(25). IST uses populations from molecular dynamics (MD) simulations on protein (solute) surfaces to calculate the cost of di...

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“…4 and SI Appendix, Fig. S8) (47). Affinities for 11 ligands were determined at least in duplicate and fit to a one-site binding model with R 2 values of at least 95%.…”

Section: Resultsmentioning

confidence: 99%

Testing inhomogeneous solvation theory in structure-based ligand discovery

Balius

Fischer

Stein

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Several cationic ligands, such as the protonated forms of 1,2-dimethyl;midazole and aniline, which were found previously to bind in the W191G mutant position") of F202G. This binding is accompanied by rearrangecavity (Fitzgerald et al, 1994(Fitzgerald et al, , 1996 at the 191 cavity, the R-factor at 2.4 8, was 17.8% (Table 1). The maximal displacement of this loop was 8.7 8, for the alpha carbon (Trp'"-Ca) and 12.6 8, for the side chain (Trp'"-CDl).…”

Section: Crystal Structures Of Ligand Trapped Conformational Changesmentioning

confidence: 94%

“…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 , 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.…”

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confidence: 99%

Protein conformer selection by ligand binding observed with crystallography

et al. 1998

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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...

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“…The plasmids for expressing the trypsin-solubilized fragment of bovine The plasmid for CcP(W191F) was obtained from Dave Goodin (The Scripps Research Institute, La Jolla, CA) (31,32). The protein was overexpressed in E. coli containing this plasmid, Intracomplex ET photocycle, simplified by ignoring association͞ dissociation of the ground and photo-excited states.…”

Section: Methodsmentioning

confidence: 99%

Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface

Hoffman

Celis²,

Cull³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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We propose that the forward and reverse halves of a flash-induced protein-protein electron transfer (ET) photocycle should exhibit differential responses to dynamic interconversion of configurations when the most stable configuration is not the most reactive, because the reactants exist in different initial configurations: the flash-photoinitiated forward ET process begins with the protein partners in an equilibrium ensemble of configurations, many of which have little or no reactivity, whereas the reactant of the thermal back ET (the charge-separated intermediate) is formed in a nonequilibrium, ''activated'' protein configuration. We report evidence for this proposal in measurements on (i) mixed-metal hemoglobin hybrids, (ii) the complex between cytochrome c peroxidase and cytochrome c, and (iii and iv) the complexes of myoglobin and isolated hemoglobin ␣-chains with cytochrome b 5. For all three systems, forward and reverse ET does respond differently to modulation of dynamic processes; further, the response to changes in viscosity is different for each system. cytochrome c ͉ dynamics ͉ hemoglobin ͉ myoglobin ͉ cytochrome c peroxidase T he long-range transfer of a single electron from donor to acceptor in a condensed phase is a fascinating and widely studied process (1, 2). Much of this work seeks to understand the electron transfer (ET) process itself. However, when the ET event involves a dynamic protein-protein interface, the observed kinetics frequently are controlled not by the ET process itself, but by the dynamics of recognition and binding and͞or conversion within an ensemble of bound configurations (3, 4).Studies of interprotein ET (3, 5-10) began with the implicit assumption of a protein-protein binding-energy landscape with a single reactive complex (Fig. 1A Left), implying a direct correlation between binding and reactivity. When the landscape for complex formation has several discrete minima ( Fig. 1 A Center) the reactive conformation may differ from the most stable one, in which case the observed ET kinetics are controlled by the rates and͞or energetics of conformational conversion within a complex (11-13). Recent studies of ET between myoglobin (Mb) and cytochrome b 5 (Fe 3ϩ b 5 ), which bind to each other by weak electrostatic interactions, disclosed a new dynamic docking paradigm in protein-protein reaction dynamics: the landscape involves numerous configurations of similar affinity, only a subset of which is active in ET (Fig. 1 A Right) (4, 14).A majority of these studies have used a photocycle in which laser-flash excitation of the metallo-porphyrin in a metalsubstituted (M ϭ Zn or Mg) hemoprotein to its triplet excited state ( 3 D) triggers ET from the triplet to the metal center of an acceptor protein (A) across a protein-protein interface, with rate constant, k f , Eq. 1:[1]The acceptor metal center of A typically is a ferri-heme center, Given the exponentially steep fall-off in the matrix element for ET between the two redox centers (19,20), there will be only a subset of conformation...

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Small Molecule Binding to an Artificially Created Cavity at the Active Site of Cytochrome c Peroxidase

Cited by 103 publications

References 48 publications

Testing inhomogeneous solvation theory in structure-based ligand discovery

Testing inhomogeneous solvation theory in structure-based ligand discovery

Protein conformer selection by ligand binding observed with crystallography

Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface

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